Method for following the progress of prostate cancer

ABSTRACT

This invention provides cDNA encoding a prostate-cancer specific marker, Repro-PC-1.0, Repro-PC-1.0 polypeptides and methods for use in diagnosis and therapy.

REFERENCE TO RELATED APPLICATION

This application is a Divisional Patent Application of Ser. No.09/680,121 filed Oct. 4, 2000, now U.S. Pat. No. 6,900,022, issued May31, 2005, which is a Continuation Patent Application of Ser. No.09/036,315 filed Mar. 6, 1998, now U.S. Pat. No. 6,218,523 B1 issuedApr. 17, 2001, which claims the benefit of the priority dates of UnitedStates provisional patent application 60/041,246, filed Mar. 7, 1997 andprovisional patent application 60/047,811 filed May 15, 1997 allincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention provides polynucleotides encoding a prostatecancer-specific cDNA, proteins encoded by the polynucleotides, andmethods of using these materials.

BACKGROUND OF THE INVENTION

The prostate is almost invariably the site of benign and malignantproliferative changes in aging males. Benign prostatic hypertrophy (BPH)is the most common non-malignant proliferative abnormality of internalorgans. A high percentage of these age-related growth disorders developinto malignancies. As a result of this, adenocarcinoma of the prostaterepresents the most common malignancy in American males and is thesecond leading cause of cancer deaths in men.

A useful method in the diagnosis of prostate cancer is determining thelevel of prostate specific antigen (PSA) in the blood. PSA is aglycoprotein secreted by the prostate gland. However, the PSA test haslimitations of sensitivity and selectivity: In general, levels above 4ng/ml are suggestive of cancer and levels above 10 ng/ml are highlysuggestive. However, many individuals with elevated levels do not haveprostate cancer, but exhibit benign prostatic hypertrophy. Conversely,many persons with prostate cancer have normal PSA levels at the time ofdiagnosis. Therefore, prostate cancer markers with greater sensitivityand selectivity for prostate cancer would be useful for, among otherthings, the diagnosis of prostate cancer.

SUMMARY OF THE INVENTION

A cDNA encoding a prostate cancer-specific marker, called Repro-PC-1.0,has been cloned and its nucleotide sequence determined. The nucleotidesequence of the cDNA and deduced amino acid sequence of Repro-PC-1.0 arepresented as SEQ ID NO:1 and SEQ ID NO:2, respectively. Repro-PC-1.0 isexpressed in prostate cancer cells and is useful as a marker in thedetection of prostate cancer. Inhibition of Repro-PC-1.0 expression isuseful in the prophylactic and therapeutic treatment of prostate cancer.

It also has been found that Repro-PC-1.0 expression is dependent onenvironment—cells from the prostate adenocarcinoma cell line, LNCaP,over-expresses Repro-PC-1.0 when propagated in male nude mice, but notwhen propagated in female nude mice. Repro-PC-1.0 has a significantlevel of amino acid sequence identity with the synaptotagmins.Therefore, it is believed that Repro-PC-1.0 functions in membrane fusionand membrane budding reactions.

Accordingly, the invention provides recombinant polynucleotide moleculescomprising a nucleotide sequence encoding Repro-PC-1.0 polypeptide orRepro-PC-1.0. Repro-PC-1.0 peptides include native Repro-PC-1.0 (SEQ IDNO:2) and allelic variants of it. Repro-PC-1.0 analogs include activeanalogs, which have the biological activity of Repro-PC-1.0, inactiveanalogs, useful as decoys, and immunogenic analogs, that, when used asan immunogen, elicit an immune response against Repro-PC-1.0 or cellsexpressing it. In one embodiment, the recombinant polynucleotidemolecule comprises a nucleotide sequence encoding at least 5 consecutiveamino acids of Repro-PC-1.0 polypeptide (SEQ ID NO:1). In anotherembodiment the nucleotide sequence is substantially identical oridentical to the nucleotide sequence of Repro-PC-1.0 (SEQ ID NO:1).

In one aspect this invention provides expression vectors comprisingexpression control sequences operatively linked to a nucleotide sequenceencoding a Repro-PC-1.0 protein or an Repro-PC-1.0 analog.

In another aspect, this invention provides polynucleotide probes andprimers of at least 7 nucleotides that specifically hybridize to asequence selected from Repro-PC-1.0 cDNA (SEQ ID NO:1) or itscomplement.

In another aspect, this invention provides an inhibitory polynucleotidecomprising an antisense sequence of at least 7 nucleotides thatspecifically hybridizes to a nucleotide sequence selected fromRepro-PC-1.0 cDNA (SEQ ID NO:1) and that inhibits expression ofRepro-PC-1.0 in cells.

In another aspect, this invention provides expression vectors comprisingexpression control sequences operably linked to a nucleotide sequenceencoding Repro-PC-1.0 polypeptide, a Repro-PC-1.0 analog or a probe,primer or inhibitory polynucleotide of this invention.

In another aspect this invention provides recombinant cells into whichhave been introduced an expression vector comprising expression controlsequences operatively linked to a nucleotide sequence encoding aRepro-PC-1.0 polypeptide or a Repro-PC-1.0 analog.

In another aspect, this invention provides a method for expressingRepro-PC-1.0 mRNA in a cell that has a nucleotide sequence encodingRepro-PC-1.0 comprising operably linking an expression control sequenceto the nucleotide sequence. The nucleotide sequence can be, for example,a sequence within the animal's genomic DNA.

In another aspect, this invention provides methods for producing aRepro-PC-1.0 polypeptide or a Repro-PC-1.0 analog comprising culturing arecombinant cell that comprises a recombinant polynucleotide thatcomprises expression control sequences operably linked to a nucleotidesequence encoding the Repro-PC-1.0 polypeptide or Repro-PC-1.0 analog.

In another aspect, this invention provides methods for detecting aRepro-PC-1.0 polynucleotide in a sample, comprising the steps of (a)contacting the sample with a polynucleotide probe or primer comprising asequence of at least 7 nucleotides that specifically hybridizes to anucleotide sequence selected from Repro-PC-1.0 and (b) detecting whetherthe polynucleotide has specifically hybridized to the Repro-PC-1.0polynucleotide. Specific hybridization provides a detection ofRepro-PC-1.0 in the sample.

In another aspect this invention provides methods for inhibitingRepro-PC-1.0 expression in a cell comprising providing the cell with aninhibitory polynucleotide of the invention or with a polynucleotide thatencodes an inactive decoy Repro-PC-1.0 analog.

In another aspect this invention provides purified, recombinantRepro-PC-1.0 protein, e.g. a protein whose amino acid sequence isidentical to the sequence of SEQ ID NO:2 and allelic variants of it. Theinvention also provides Repro-PC-1.0 analogs whose amino acid sequenceis not naturally occurring and is substantially identical to the aminoacid sequence of Repro-PC-1.0 (SEQ ID NO:2). Such analogs include activeanalogs having the biological activity of Repro-PC-1.0, as well asimmunogenic analogs, capable of eliciting the production of antibodiesthat recognize Repro-PC-1.0.

In another aspect, this invention provides antibodies that specificallybind to Repro-PC-1.0 polypeptide.

In another aspect, this invention provides methods for detecting aRepro-PC-1.0 polypeptide in a sample, comprising the steps of (a)contacting the sample with an antibody that specifically binds to theRepro-PC-1.0 polypeptide and (b) detecting specific binding between theantibody and Repro-PC-1.0 polypeptide. Specific binding provides adetection of Repro-PC-1.0 in the sample.

In another aspect, this invention provides methods for diagnosing,monitoring or making a prognosis for prostate cancer in a subject. Themethods involve detecting Repro-PC-1.0 mRNA or polypeptide in a samplefrom the subject. In the diagnostic method, a diagnostic amount ofRepro-PC-1.0 mRNA or Repro-PC-1.0 polypeptide in a sample from thesubject is determined and the diagnostic amount is compared with anormal range of Repro-PC-1.0 mRNA or Repro-PC-1.0 polypeptide in anon-cancerous control sample. A diagnostic amount above the normal rangeprovides a positive indication in the diagnosis of prostate cancer. Thedetection of an amount of Repro-PC-1.0 mRNA or polypeptide at aparticular prognostic level provides a prognosis for the subject.Methods for monitoring the progress of prostate cancer involve detectingthe amount of Repro-PC-1.0 mRNA or Repro-PC-1.0 polypeptide in thesubject at a first and a second time, and comparing the amounts. Achange in the amount indicates a change in the course of the disease,with a decreasing amount indicating remission of prostate cancer andincrease indicating progression of the prostate cancer. One embodimentof these methods involves diagnostic imaging of Repro-PC-1.0 in the bodyusing detectably labeled probes, primers or antibodies. Methods formaking a prognosis for prostate cancer in a subject involve determiningthe amount of Repro-PC-1.0 polynucleotide or polypeptide in a subjectsample and comparing that amount to a prognostic amount. A determinedamount at a particular prognostic amount provides a prognosis for thesubject.

In another aspect, this invention provides a method of detecting achromosomal translocation of a Repro-PC-1.0 gene comprising the steps ofhybridizing a labeled probe of the invention to a chromosome spread froma cell sample to determine the pattern of hybridization and determiningwhether the pattern of hybridization differs from a normal pattern.

In another aspect, this invention provides methods for the prophylacticor therapeutic treatment of prostate cancer in a subject. One methodinvolves providing prostate cancer cells with an inhibitorypolynucleotide of the invention. Another method involves administeringto the subject a composition comprising antibodies that specificallybind to Repro-PC-1.0 coupled to a toxin that kills or inhibits thegrowth of prostate cancer cells. Another method involves altering thehormonal environment of the cancer cells to suppress expression ofRepro-PC-1.0.

In another aspect, this invention provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and pharmacologicallyeffective amount of an inhibitory polynucleotide of the invention or atoxin-conjugated antibody of the invention.

In another aspect, this invention provides polynucleotide andpolypeptide vaccines for eliciting a humoral or cell-mediated immuneresponse against Repro-PC-1.0 or cells expressing it. A polypeptide orpolynucleotide vaccine for eliciting an immune response againstRepro-PC-1.0 comprises an immunogenic Repro-PC-1.0 polypeptide analog ora polynucleotide encoding the analog. In one embodiment, the immunogenicanalog bears an MHC Class I or MHC Class II binding motif.

In another aspect, this invention provides a method for eliciting in asubject an immune response against a cell bearing Repro-PC-1.0 on itssurface comprising administering to the subject a vaccine of theinvention. One method for eliciting an MHC Class I-dependentcell-mediated immune response involves providing the subject with cellstransfected with an expression vector that encodes Repro-PC-1.0polypeptide, or Repro-PC-1.0 analogs comprising amino acid motifsrecognized by MHC Class I molecules.

In another aspect, this invention provides screening methods fordetermining whether a compound modulates (inhibits or promotes) theexpression of Repro-PC-1.0 in a cell. The methods involve contacting thecell with the compound and determining whether the production ofRepro-PC-1.0 mRNA or polypeptide are altered (increased, decreased orunchanged) in a statistically significant manner (p<0.05).

In another aspect, this invention provides screening methods fordetermining whether a compound inhibits the activity of Repro-PC-1.0.The methods involve contacting a cell expressing Repro-PC-1.0 with thecompound and determining whether exocytotic events have been altered.According to one method, a determination is made as to whether theamount or character of secretions by the cell into the medium is alteredby the compound. In another method, the compound is tested for itsability to alter the capacitance across the cell membrane.

In another aspect this invention provides methods for detectingpolymorphic forms of Repro-PC-1.0 comprising comparing the identity of anucleotide or amino acid at a selected position from the sequence of atest Repro-PC-1.0 gene or polypeptide with identity of the nucleotide oramino acid at the corresponding position of native Repro-PC-1.0 (SEQ IDNO:1 or 2). A difference in identity indicates that the testpolynucleotide is a polymorphic form of Repro-PC-1.0.

In another aspect, this invention provides a transgenic non-humananimal, preferably a mammal, whose germ cells comprise a recombinantpolynucleotide of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are photographs of LNCaP cells grown in male (1A) and female(1B) mice.

FIGS. 2A-2B are photographs of a Northern analysis. FIG. 2A shows RNAfrom LNCaP cells and hybridized with Repro-PC-1.0-specific probe. FIG.2B shows rehybridization of the same blot with probes for tubulin andactin. Male=male-grown tumors. Female=female-grown tumors. C=LNCaPcells. P=PC3 cells.

FIG. 3 shows the alignment of overlapping clones which, together,produce the Repro-PC-1.0 cDNA of SEQ ID NO:1.

FIG. 4 presents a comparison of the organization of PKC, Repro-PC-1.0and synaptotagmin polypeptides.

FIG. 5 shows the alignment of the amino acid sequences of Repro-PC-1.0(SEQ ID NO:2) and rat synaptotagmin 4 (“SYT4”) (SEQ ID NO:5).

FIG. 6 shows the alignment of the internal repeats of PKC-C2 (SEQ IDNO:6), Repro-PC-1.0 “B” repeat (SEQ ID NO:7), synaptotagmin “B” repeat(SEQ ID NO:8), synaptotagmin “A” repeat (SEQ ID NO:9) and Repro-PC-1.0“A” repeat (SEQ ID NO:10).

DETAILED DESCRIPTION

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2d ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); and Hale & Marham, The Harper CollinsDictionary of Biology (1991). Although any methods and materials similaror equivalent to those described herein can be used in the practice ortesting of the present invention, preferred methods and materials aredescribed. As used herein, the following terms have the meaningsascribed to them unless specified otherwise.

“Polynucleotide” refers to a polymer composed of nucleotide units(ribonucleotides, deoxyribonucleotides, related naturally occurringstructural variants, and synthetic non-naturally occurring analogsthereof) linked via phosphodiester bonds, related naturally occurringstructural variants, and synthetic non-naturally occurring analogsthereof. Thus, the term includes nucleotide polymers in which thenucleotides and the linkages between them include non-naturallyoccurring synthetic analogs, such as, for example and withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleicacids (PNAs), and the like. Such polynucleotides can be synthesized, forexample, using an automated DNA synthesizer. The term “nucleic acid”typically refers to large polynucleotides. The term “oligonucleotide”typically refers to short polynucleotides, generally no greater thanabout 50 nucleotides. It will be understood that when a nucleotidesequence is represented by a DNA sequence (i.e., A, T, G, C), this alsoincludes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction. Thedirection of 5′ to 3′ addition of nucleotides to nascent RNA transcriptsis referred to as the transcription direction. The DNA strand having thesame sequence as an mRNA is referred to as the “coding strand”;sequences on the DNA strand having the same sequence as an mRNAtranscribed from that DNA and which are located 5′ to the 5′-end of theRNA transcript are referred to as “upstream sequences”; sequences on theDNA strand having the same sequence as the RNA and which are 3′ to the3′ end of the coding RNA transcript are referred to as “downstreamsequences.”

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.The term “protein” typically refers to large polypeptides. The term“peptide” typically refers to short polypeptides.

“Naturally-occurring” as applied to an object refers to the fact thatthe object can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory isnaturally-occurring.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

A “reference sequence” is a defined sequence used as a basis for asequence comparison and may be a subset of a larger sequence, e.g., acomplete cDNA, protein, or gene sequence.

Because two polynucleotides or polypeptides each may comprise (1) asequence (i.e., only a portion of the complete polynucleotide orpolypeptide sequence) that is similar between the two polynucleotides,or (2) a sequence that is divergent between the two polynucleotides,sequence comparisons between two (or more) polynucleotides orpolypeptides are typically performed by comparing sequences of the twopolynucleotides over a “comparison window” to identify and compare localregions of sequence similarity.

A “comparison window” refers to a conceptual segment of typically atleast 12 consecutive nucleotide or 4 consecutive amino acid residuesthat is compared to a reference sequence. The comparison windowfrequently has a length of at least 15 or at least 25 nucleotides or atleast 5 or at least 8 amino acids. The comparison window may compriseadditions or deletions (i.e., gaps) of about 20 percent or less ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. Optimal alignmentof sequences for aligning a comparison window may be conducted bycomputerized implementations of algorithms (GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package Release 7.0, GeneticsComputer Group, 575 Science Dr., Madison, Wis.) or by inspection, andthe best alignment (i.e., resulting in the highest percentage ofhomology over the comparison window) generated by any of the variousmethods is selected.

A subject nucleotide sequence or amino acid sequence is “identical” to areference sequence if the two sequences are the same when aligned formaximum correspondence over the length of the nucleotide or amino acidsequence.

The “percentage of sequence identity” between two sequences iscalculated by comparing two optimally aligned sequences over acomparison window, determining the number of positions at which theidentical nucleotide or amino acid occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity. Unless otherwise specified, the comparison windowused to compare two sequences is the length of the shorter sequence.

Alternatively, when percentage of sequence identity is used in referenceto polypeptides it is recognized that residue positions that are notidentical often differ by conservative amino acid substitutions, whereamino acids residues are substituted for other amino acid residues withsimilar chemical properties (e.g., charge or hydrophobicity) andtherefore do not change the functional properties of the molecule. Wheresequences differ in conservative substitutions, the percent sequenceidentity may be adjusted upwards to correct for the conservative natureof the substitution. Means for making this adjustment are well known tothose of skill in the art. Typically this involves scoring aconservative substitution as a partial rather than a full mismatch,thereby increasing the percentage sequence identity. Thus, for example,where an identical amino acid is given a score of 1 and anon-conservative substitution is given a score of zero, a conservativesubstitution is given a score between zero and 1. The scoring ofconservative substitutions is calculated, e.g., according to knownalgorithm. See, e.g., Meyers & Miller, Computer Applic. Biol. Sci., 4:11-17 (1988); Smith & Waterman, Adv. Appl. Math. 2: 482 (1981);Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970); Pearson & Lipman,Proc. Natl. Acad. Sci. USA 85: 2444 (1988); Higgins & Sharp Gene, 73:237-244 (1988); Higgins & Sharp, CABIOS 5: 151-153 (1989); Corpet etal., Nucleic Acids Research 16: 10881-90 (1988); Huang et al., ComputerApplications in the Biosciences 8: 155-65 (1992); and Pearson et al.,Methods in Molecular Biology 24: 307-31 (1994). Alignment is also oftenperformed by inspection and manual alignment.

A subject nucleotide sequence or amino acid sequence is “substantiallyidentical” to a reference sequence if the subject amino acid sequence ornucleotide sequence has at least 90% sequence identity over a comparisonwindow. Thus, sequences that have at least 95% sequence identity, atleast 98% sequence identity or at least 99% sequence identity with thereference sequence are also “substantially identical.” Two sequencesthat are identical to each other are, of course, also “substantiallyidentical”.

“Complementary” refers to the topological compatibility or matchingtogether of interacting surfaces of two polynucleotides. Thus, the twomolecules can be described as complementary, and furthermore, thecontact surface characteristics are complementary to each other. A firstpolynucleotide is complementary to a second polynucleotide if thenucleotide sequence of the first polynucleotide is identical to thenucleotide sequence of the polynucleotide binding partner of the secondpolynucleotide. Thus, the polynucleotide whose sequence 5′-TATAC-3′ iscomplementary to a polynucleotide whose sequence is 5′-GTATA-3′.

A nucleotide sequence is “substantially complementary” to a referencenucleotide sequence if the sequence complementary to the subjectnucleotide sequence is substantially identical to the referencenucleotide sequence.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Expression control sequence” refers to a nucleotide sequence in apolynucleotide that regulates the expression (transcription and/ortranslation) of a nucleotide sequence operatively linked thereto.“Operatively linked” refers to a functional relationship between twoparts in which the activity of one part (e.g., the ability to regulatetranscription) results in an action on the other part (e.g.,transcription of the sequence). Expression control sequences caninclude, for example and without limitation, sequences of promoters(e.g., inducible or constitutive), enhancers, transcription terminators,a start codon (i.e., ATG), splicing signals for introns, and stopcodons.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in vitro expressionsystem. Expression vectors include all those known in the art, such ascosmids, plasmids (e.g., naked or contained in liposomes) and virusesthat incorporate the recombinant polynucleotide.

“Conservative substitution” refers to the substitution in a polypeptideof an amino acid with a functionally similar amino acid. The followingsix groups each contain amino acids that are conservative substitutionsfor one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

“Allelic variant” refers to any of two or more polymorphic forms of agene occupying the same genetic locus. Allelic variations arisenaturally through mutation, and may result in phenotypic polymorphismwithin populations. Gene mutations can be silent (no change in theencoded polypeptide) or may encode polypeptides having altered aminoacid sequences. “Allelic variants” also refer to cDNAs derived from mRNAtranscripts of genetic allelic variants, as well as the proteins encodedby them.

“Recombinant polynucleotide” refers to a polynucleotide having sequencesthat are not naturally joined together. An amplified or assembledrecombinant polynucleotide may be included in a suitable vector, and thevector can be used to transform a suitable host cell. A host cell thatcomprises the recombinant polynucleotide is referred to as a“recombinant host cell.” The gene is then expressed in the recombinanthost cell to produce, e.g., a “recombinant polypeptide.” A recombinantpolynucleotide may serve a non-coding function (e.g., promoter, originof replication, ribosome-binding site, etc.) as well.

“Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, whichspecifically bind and recognize an analyte (antigen). The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Antibodies exist, e.g., as intactimmunoglobulins or as a number of well characterized fragments producedby digestion with various peptidases. This includes, e.g., Fab′ andF(ab)′₂ fragments. The term “antibody,” as used herein, also includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies.

“Immunoassay” refers to an assay that utilizes an antibody tospecifically bind an analyte. The immunoassay is characterized by theuse of specific binding properties of a particular antibody to isolate,target, and/or quantify the analyte.

“Vaccine” refers to an agent or composition containing an agenteffective to confer a therapeutic degree of immunity on an organismwhile causing only very low levels of morbidity or mortality. Methods ofmaking vaccines are, of course, useful in the study of the immune systemand in preventing and treating animal or human disease.

An “immunogenic amount” is an amount effective to elicit an immuneresponse in a subject.

“Hybridizing specifically to” or “specific hybridization” or“selectively hybridize to”, refers to the binding, duplexing, orhybridizing of a nucleic acid molecule preferentially to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA.

The term “stringent conditions” refers to conditions under which a probewill hybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences. “Stringent hybridization”and “stringent hybridization wash conditions” in the context of nucleicacid hybridization experiments such as Southern and northernhybridizations are sequence dependent, and are different under differentenvironmental parameters. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I chapter 2 “Overview of principles of hybridization and thestrategy of nucleic acid probe assays”, Elsevier, N.Y. Generally, highlystringent hybridization and wash conditions are selected to be about 5°C. lower than the thermal melting point (Tm) for the specific sequenceat a defied ionic strength and pH. The Tm is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. Very stringent conditions areselected to be equal to the Tm for a particular probe.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of highly stringent wash conditions is 0.15 M NaClat 72° C. for about 15 minutes. An example of stringent wash conditionsis a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook et al. for adescription of SSC buffer). Often, a high stringency wash is preceded bya low stringency wash to remove background probe signal. An examplemedium stringency wash for a duplex of, e.g., more than 100 nucleotidesis 1×SSC at 45° C. for 15 minutes. An example low stringency wash for aduplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15minutes. In general, a signal to noise ratio of 2× (or higher) than thatobserved for an unrelated probe in the particular hybridization assayindicates detection of a specific hybridization.

An antibody “specifically binds to” or “is specifically immunoreactivewith” a protein when the antibody functions in a binding reaction whichis determinative of the presence of the protein in the presence of aheterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified antibodies bindpreferentially to a particular protein and do not bind in a significantamount to other proteins present in the sample. Specific binding to aprotein under such conditions requires an antibody that is selected forits specificity for a particular protein. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select monoclonal antibodies specificallyimmunoreactive with a protein. See Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York, for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity.

A first sequence is an “antisense sequence” with respect to a secondsequence if a polynucleotide whose sequence is the first sequencespecifically hybridizes with a polynucleotide whose sequence is thesecond sequence.

“Substantially pure” means an object species is the predominant speciespresent (i.e., on a molar basis, more abundant than any other individualmacromolecular species in the composition), and a substantially purifiedfraction is a composition wherein the object species comprises at leastabout 50% (on a molar basis) of all macromolecular species present.Generally, a substantially pure composition means that about 80% to 90%or more of the macromolecular species present in the composition is thepurified species of interest. The object species is purified toessential homogeneity (contaminant species cannot be detected in thecomposition by conventional detection methods) if the compositionconsists essentially of a single macromolecular species. Solventspecies, small molecules (<500 Daltons), stabilizers (e.g., BSA), andelemental ion species are not considered macromolecular species forpurposes of this definition.

“Primer” refers to a polynucleotide that is capable of specificallyhybridizing to a designated polynucleotide template and providing apoint of initiation for synthesis of a complementary polynucleotide.Such synthesis occurs when the polynucleotide primer is placed underconditions in which synthesis is induced, i.e., in the presence ofnucleotides, a complementary polynucleotide template and an agent forpolymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with, e.g.,chromogenic, radioactive, or fluorescent moieties and used as detectablemoieties.

“Probe” refers to a polynucleotide that is capable of specificallyhybridizing to a designated sequence of another polynucleotide. A probespecifically hybridizes to a target complementary polynucleotide, butneed not reflect the exact complementary sequence of the template. Insuch a case, specific hybridization of the probe to the target dependson the stringency of the hybridization conditions. Probes can be labeledwith, e.g., chromogenic, radioactive, or fluorescent moieties and usedas detectable moieties.

“Detecting” refers to determining the presence, absence, or amount of ananalyte in a sample, and can include quantifying the amount of theanalyte in a sample or per cell in a sample.

“Detectable moiety” or a “label” refers to a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, ³⁵S, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin-streptavadin, dioxigenin, haptens and proteins for which antiseraor monoclonal antibodies are available, or nucleic acid molecules with asequence complementary to a target. The detectable moiety oftengenerates a measurable signal, such as a radioactive, chromogeric, orfluorescent signal that can be used to quantitate the amount of bounddetectable moiety in a sample. The detectable moiety can be incorporatedin or attached to a primer or probe either covalently, or through ionic,van der Waals or hydrogen bonds, e.g., incorporation of radioactivenucleotides, or biotinylated nucleotides that are recognized bystreptavadin. The detectable moiety may be directly or indirectlydetectable. Indirect detection can involve the binding of a seconddirectly or indirectly detectable moiety to the detectable moiety. Forexample, the detectable moiety can be the ligand of a binding partner,such as biotin, which is a binding partner for streptavadin, or anucleotide sequence, which is the binding partner for a complementarysequence, to which it can specifically hybridize. The binding partnermay itself be directly detectable, for example, an antibody may beitself labeled with a fluorescent molecule. The binding partner also maybe indirectly detectable, for example, a nucleic acid having acomplementary nucleotide sequence can be a part of a branched DNAmolecule that is in turn detectable through hybridization with otherlabeled nucleic acid molecules. (See, e.g., P D. Fahrlander and A.Klausner, Bio/Technology (1988) 6:1165.) Quantitation of the signal isachieved by, e.g., scintillation counting, densitometry, or flowcytometry.

“Linker” refers to a molecule that joins two other molecules, eithercovalently, or through ionic, van der Waals or hydrogen bonds, e.g., anucleic acid molecule that hybridizes to one complementary sequence atthe 5′ end and to another complementary sequence at the 3′ end, thusjoining two non-complementary sequences.

“Amplification” refers to any means by which a polynucleotide sequenceis copied and thus expanded into a larger number of polynucleotidemolecules, e.g., by reverse transcription, polymerase chain reaction,and ligase chain reaction.

“Prognostic value” refers to an amount of an analyte in a subject samplethat is consistent with a particular prognosis for a designated disease.The amount (including a zero amount) of the analyte detected in a sampleis compared to the prognostic value for the sample such that therelative comparison of the values indicates the likely outcome of theprogression of the disease.

“Diagnostic value” refers to a value that is determined for an analytein a subject sample, which is then compared to a normal range of theanalyte in a sample. (e.g., from a healthy individual) such that therelative comparison of the values provides a reference value fordiagnosing a designated disease. Depending upon the method of detection,the diagnostic value may be a determination of the amount of theanalyte, but it is not necessarily an amount. The diagnostic value mayalso be a relative value, such as a plus or a minus score and alsoincludes a value indicating the presence or absence of the analyte in asample.

“Pharmaceutical composition” refers to a composition suitable forpharmaceutical use in a mammal. A pharmaceutical composition comprises apharmacologically effective amount of an active agent and apharmaceutically acceptable carrier. “Pharmacologically effectiveamount” refers to that amount of an agent effective to produce theintended pharmacological result. “Pharmaceutically acceptable carrier”refers to any of the standard pharmaceutical carriers, buffers, andexcipients, such as a phosphate buffered saline solution, 5% aqueoussolution of dextrose, and emulsions, such as an oil/water or water/oilemulsion, and various types of wetting agents and/or adjuvants. Suitablepharmaceutical carriers and formulations are described in Remington'sPharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995).Preferred pharmaceutical carriers depend upon the intended mode ofadministration of the active agent. Typical modes of administrationinclude enteral (e.g., oral) or parenteral (e.g., subcutaneous,intramuscular, or intravenous intraperitoneal injection; or topical,transdermal, or transmucosal administration).

A “subject” of diagnosis or treatment is an animal, such as a mammal,including a human. Non-human animals subject to treatment include, forexample, fish, birds, and mammals such as primates, cows, sheep, pigs,horses, dogs and cats.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing pathology.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

II. cDNA Encoding Repro-PC-1.0

A cDNA molecule encoding a prostate cancer-specific marker, calledRepro-PC-1.0, has been isolated. The nucleotide sequence and deducedamino acid sequence of the nucleic acid molecule are presented in SEQ IDNO:1 and SEQ ID NO:2, respectively. This 3891-base nucleotide sequencecontains an open reading frame of 1275 bases encoding Repro-PC-1.0 fromnucleotide 151 to nucleotide 1425. The deduced amino acid sequence ofRepro-PC-1.0 has 425 amino acids.

Analysis of the deduced amino acid sequence of Repro-PC-1.0 shows 90%identity with rat synaptotagmin IV. Amino acid residues 15-37 havesufficient length and hydrophobicity to constitute a transmembranedomain that displays the unusual transmembrane boundaries of othersynaptotagmins. Repro-PC-1.0 also has two copies of a 116 amino aciddirect repeat (amino acids 150-252 and 276-396) that have 34% identitywith each other. These repeats are homologous to the C2 regulatorydomain of calcium-dependent isoforms of protein kinase C (PKC) and otherisoforms of the synaptotagmins.

The synaptotagmins are synaptic vesicle proteins proposed to play a rolein regulating synaptic vesicle translocation to the presynaptic releasesite of the plasma membrane (docking) and/or fusion of these twomembranes. Therefore, while not wishing to be limited by theory, it isbelieved that Repro-PC-1.0 functions in exocytosis and endocytosispathways.

III. Repro-PC-1.0 Nucleic Acids

Accordingly, this invention provides recombinant polynucleotidescomprising nucleotide sequences encoding Repro-PC-1.0 proteins,Repro-PC-1.0 analogs or fragments of them, as described herein. Analogsinclude “active analogs” having the biological activity of Repro-PC-1.0,“inactive analogs” useful, e.g., as decoys, and “immunogenic analogs,”which, when presented as an immunogen, elicit the production of anantibody which specifically binds to Repro-PC-1.0. The polynucleotidesare useful for expressing the mRNA or polypeptides they encode and inthe preparation of probes or primers, among other things.

In one embodiment, the recombinant polynucleotide molecule comprises anucleotide sequence encoding a sequence of at least 5 amino acidsselected from the amino acid sequence of Repro-PC-1.0 (SEQ ID NO:2). Thenucleotide sequence can encode a sequence of at least 25 amino acids, atleast 100 amino acids or at least 200 amino acids from SEQ ID NO:2. Inone embodiment, the nucleotide sequence encodes full-length nativeRepro-PC-1.0 polypeptide.

The nucleotide sequence can be identical to a sequence from Repro-PC-1.0cDNA or its complement, or can include degenerate codons. In oneembodiment of a nucleotide sequence encoding full-length Repro-PC-1.0,the sequence is identical to the coding sequence of Repro-PC-1.0 of SEQID NO:1. In another embodiment the nucleotide sequence encodes aRepro-PC-1.0 analog whose amino acid sequence is substantially identicalto the amino acid sequence of Repro-PC-1.0 polypeptide (SEQ ID NO:2).

In another embodiment, the polynucleotide encodes a fusion proteinbetween Repro-PC-1.0 polypeptide or Repro-PC-1.0 analog amino acidsequences and a second amino acid sequence.

The polynucleotides of the present invention are cloned or amplified byin vitro methods, such as the polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR) andthe Qβ replicase amplification system (QB). For example, apolynucleotide encoding the protein can be isolated by polymerase chainreaction of cDNA from prostate cancer cells using primers based on theDNA sequence of Repro-PC-1.0 of SEQ ID NO:1. One pair of primers usefulfor amplifying Repro-PC-1.0 DNA, including allelic variants, is:

-   5′ oligo (109)    Upper Primer, 21-mer, position 109:

5′ CAG TTT TCC CTT CAG CAC CTC 3′ (SEQ ID NO:3)

-   3′ oligo (3489):    Lower Primer, 30-mer, position 3489:

5′ TTC CTT TGT TGT TTC TTT TCT CTT (SEQ ID NO:4) TTC TGA 3′

A wide variety of cloning and in vitro amplification methodologies arewell-known to persons of skill. PCR methods are described in, forexample, U.S. Pat. No. 4,683,195; Mullis et al. (1987) Cold SpringHarbor Symp. Quant. Biol. 51:263; and Erlich, ed., PCR Technology,(Stockton Press, NY, 1989). Polynucleotides also can be isolated byscreening genomic or cDNA libraries with probes selected from thesequences of SEQ ID NO:1 under stringent hybridization conditions.

Mutant versions of the proteins can be made by site-specific mutagenesisof other polynucleotides encoding the proteins, or by random mutagenesiscaused by increasing the error rate of PCR of the originalpolynucleotide with 0.1 mM MnCl₂ and unbalanced nucleotideconcentrations.

This invention also provides expression vectors. e.g., recombinantpolynucleotide molecules comprising expression control sequencesoperatively linked to a nucleotide sequence encoding the targetpolypeptide. Expression vectors can be adapted for function inprokaryotes or eukaryotes by inclusion of appropriate promoters,replication sequences, markers, etc. for transcription and translationof mRNA. The construction of expression vectors and the expression ofgenes in transfected cells involves the use of molecular cloningtechniques also well known in the art. Sambrook et al., MolecularCloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., (1989) and Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., (Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc.) Usefulpromoters for such purposes include a metallothionein promoter, aconstitutive adenovirus major late promoter, a dexamethasone-inducibleMMTV promoter, a SV40 promoter, a MRP polIII promoter, a constitutiveMPSV promoter, a tetracycline-inducible CMV promoter (such as the humanimmediate-early CMV promoter), and a constitutive CMV promoter. Aplasmid useful for gene therapy can comprise other functional elements,such as selectable markers, identification regions, and other genes.Recombinant DNA expression plasmids can also be used to prepare thepolynucleotides of the invention for delivery by means other than bygene therapy, although it may be more economical to make shortoligonucleotides by in vitro chemical synthesis.

Methods for transfecting genes into mammalian cells and obtaining theirexpression for in vitro use or for gene therapy, are well known to theart. See, e.g., Methods in Enzymology, vol. 185, Academic Press, Inc.,San Diego, Calif. (D. V. Goeddel, ed.) (1990) or M. Krieger, GeneTransfer and Expression—A Laboratory Manual, Stockton Press, New York,N.Y., (1990).

Expression vectors useful in this invention depend on their intendeduse. Such expression vectors must, of course, contain expression andreplication signals compatible with the host cell. Expression vectorsuseful for expressing the protein of this invention include viralvectors such as alpha viruses, retroviruses, adenoviruses andadeno-associated viruses, plasmid vectors, cosmids, liposomes and thelike. Viral and plasmid vectors are preferred for transfecting mammaliancells. The expression vector pcDNA1 (Invitrogen, San Diego, Calif.), inwhich the expression control sequence comprises the CMV promoterprovides good rates of transfection and expression. Adeno-associatedviral vectors are useful in the gene therapy methods of this invention.

The construct can also contain a tag to simplify isolation of theprotein. For example, a polyhistidine tag of, e.g., six histidineresidues, can be incorporated at the amino terminal end of the protein.The polyhistidine tag allows convenient isolation of the protein in asingle step by nickel-chelate chromatography.

In another embodiment, endogenous genes are transcribed by operativelylinking them to expression control sequences supplied endogenously thatrecombine with genomic DNA. In one method, one provides the cell with arecombinant polynucleotide containing a targeting sequence, whichpermits homologous recombination into the genome upstream of thetranscriptional start site of target gene; the expression controlsequences; an exon of the target gene; and an unpaired splice-donor sitewhich pairs with a splice acceptor in the target gene. Such methods arediscussed in Treco et al., WO 94/12650; Treco et al., WO 95/31560 andTreco et al., WO 96/29411.

The invention also provides recombinant cells comprising an expressionvector for expression of the nucleotide sequences encoding a polypeptideof this invention. Host cells can be selected for high levels ofexpression in order to purify the protein. Mammalian cells are preferredfor this purpose, but prokaryotic cells, such as E. coli, also areuseful. The cell can be, e.g., a recombinant cell in culture or a cellin vivo.

IV. Polynucleotide Probes and Primers

This invention provides polynucleotide probes and primers thatspecifically hybridize to a subsequence of Repro-PC-1.0 cDNA or itscomplement under stringent hybridization conditions. The probes andprimers of this invention are polynucleotides of at least 7 nucleotides,at least 10 nucleotides, at least 15 nucleotides, at least 20nucleotides or at least 25 nucleotides. In one embodiment, the sequenceof the polynucleotide is a contiguous sequence from SEQ ID NO:1 or itscomplement. Any suitable region of the Repro-PC-1.0 gene may be chosenas a target for polynucleotide hybridization. Nucleotide substitutions,deletions, and additions may be incorporated into the polynucleotides aslong as the characteristic ability to specifically hybridize to thetarget sequence or its complement is retained. Nucleotide sequenceVariation may result from sequence polymorphisms of various allelesminor sequencing errors, and the like.

The probes and primers of the invention are useful as probes inhybridization assays, such as Southern and Northern blots foridentifying polynucleotides having a nucleotide sequence encoding aRepro-PC-1.0 polypeptide, and as primers for amplification procedures.The probes and primers of the invention are also useful in detecting thepresence, absence or amount of Repro-PC-1.0 in tissue biopsies andhistological sections where the detection method is carried out in situ,typically after amplification of Repro-PC-1.0 sequences using a primerset.

The probes and primers of this invention also are useful for identifyingallelic forms of Repro-PC-1.0 and animal cognate genes. Probes andprimers can be used to screen human or animal genomic DNA or cDNAlibraries under, e.g., stringent conditions. DNA molecules thatspecifically hybridize to the probe are then further examined todetermine whether they are Repro-PC-1.0 allelic variants or animalcognates.

The probes also are useful in oligonucleotide arrays. Such arrays areused in hybridization assays to check the identity of bases in a targetpolynucleotide. In essence, when a target hybridizes perfectly to aprobe on the array, the target contains the nucleotide sequence of theprobe. When the target hybridizes less well, or does not hybridize atall, then the target and probe differ in sequence by one or morenucleotide. By proper selection of probes, one can check bases on atarget molecule. See, e.g., Chee et al., WO 95/11995. The use theRepro-PC-1.0 sequence in genomics is described further below.

In one embodiment, the polynucleotide further comprises a label. Adetectable moiety bound to either an oligonucleotide primer or a probeis subsequently used to detect hybridization of an oligonucleotideprimer to the RNA component. Detection of labeled material bound to aRepro-PC-1.0 polynucleotide in a sample provides a means of determininga diagnostic or prognostic value.

Although primers and probes can differ in sequence and length, theprimary differentiating factor is one of function: primers serve as aninitiation point for DNA synthesis of a target polynucleotide, as in RTand PCR reactions, while probes are typically used for hybridization toand detection of a target polynucleotide. Typical lengths of primers orprobes can range from 7-50 nucleotides, preferably from 10-40nucleotides, and most preferably from 15-35 nucleotides. A primer orprobe can also be labeled with a detectable moiety for detection ofhybridization of the primer or probe to the target polynucleotide.

In general, those of skill in the art recognize that the polynucleotidesused in the invention include both DNA and RNA molecules and naturallyoccurring modifications thereof, as well as synthetic, non-naturallyoccurring analogs of the same, and heteropolymers, ofdeoxyribonucleotides, ribonucleotides, and/or analogues of either. Theparticular composition of a polynucleotide or polynucleotide analog willdepend upon the purpose for which the material will be used and theenvironment in which the material will be placed. Modified or synthetic,non-naturally occurring nucleotides have been designed to serve avariety of purposes and to remain stable in a variety of environments,such as those in which nucleases are present.

Oligonucleotides preferably are synthesized, e.g., on an AppliedBioSystems or other commercially available oligonucleotide synthesizeraccording to specifications provided by the manufacturer.Oligonucleotides may be prepared using any suitable method, such as thephosphotriester and phosphodiester methods, or automated embodimentsthereof. In one such automated embodiment, diethylphosphoramidates areused as starting materials and may be synthesized as described byBeaucage et al., Tetrahedron Letters 22; 1859 (1981), and U.S. Pat. No.4,458,066.

Polynucleotides, e.g., probes, also can be recombinantly producedthrough the use of plasmids or other vectors.

V. Methods for Detecting Repro-PC-1.0 Polynucleotides

The probes and primers of this invention are useful, among other things,in detecting Repro-PC-1.0 polynucleotides in a sample. A method fordetecting the presence, absence or amount of a Repro-PC-1.0polynucleotide in a sample involves two steps: (1) specificallyhybridizing a polynucleotide probe or primer to a Repro-PC-1.0polynucleotide, and (2) detecting the specific hybridization.

For the first step of the method, the polynucleotide used for specifichybridization is chosen to hybridize to any suitable region ofRepro-PC-1.0. The polynucleotide can be a DNA or RNA molecule, as wellas a synthetic, non-naturally occurring analog of the same. Thepolynucleotides in this step are polynucleotide primers andpolynucleotide probes disclosed herein.

For the second step of the reaction, any suitable method for detectingspecific hybridization of a polynucleotide to Repro-PC-1.0 may be used.Such methods include, e.g., amplification by extension of a hybridizedprimer using reverse transcriptase (RT); extension of a hybridizedprimer using RT-PCR or other methods of amplification; and in situdetection of a hybridized primer. In in situ hybridization, a sample oftissue or cells is fixed onto a glass slide and permeablizedsufficiently for use with in situ hybridization techniques. Detectablemoieties used in these methods include, e.g., labeled polynucleotideprobes; direct incorporation of label in amplification or RT reactions,and labeled polynucleotide primers.

Often, cell extracts or tissue samples used in methods for determiningthe amount of a polynucleotide in a sample will contain variable amountsof cells or extraneous extracellular matrix materials. Thus, a methodfor determining the cell number in a sample is important for determiningthe relative amount per cell of a test polynucleotide such asRepro-PC-1.0. A control for cell number and amplification efficiency isuseful for determining diagnostic values for a sample of a potentialcancer, and a control is particularly useful for comparing the amount oftest polynucleotide such as Repro-PC-1.0 in sample to a prognostic valuefor prostate cancer. A preferred embodiment of the control RNA isendogenously expressed 28S rRNA. (See, e.g., Khan et al., Neurosci.Lett. 147: 114-117 (1992) which used 28S rRNA as a control, by dilutingreverse transcribed 28S rRNA and adding it to the amplificationreaction.)

VI. Inhibitory Polynucleotides for Inhibiting Repro-PC-1.0 Expression

A. General

This invention also provides inhibitory polynucleotides directed againstRepro-PC-1.0 polynucleotides that inhibit Repro-PC-1.0 expression and,therefore inhibit its activity in a cell. Inhibitory polynucleotides caninhibit Repro-PC-1.0 activity in a number of ways. According to onemechanism, the polynucleotide prevents transcription of the Repro-PC-1.0gene (for instance, by triple helix formation). In another mechanism,the polynucleotide destabilizes the Repro-PC-1.0 and reduces itshalf-life. In another mechanism the polynucleotide inhibits assembly ofthe RNA component into the Repro-PC-1.0 by binding to Repro-PC-1.0.

An inhibitory polynucleotide is a polynucleotide that is capable ofspecifically hybridizing with a target polynucleotide and thatinterferes with the transcription, processing, translation or otheractivity the target polynucleotide. Inhibitory polynucleotides generallyare single-stranded and have a sequence of at least 7, 8, 9, 10, or 11nucleotides capable of specifically hybridizing to the target sequence.RNA sequences generally require a sequence of at least 10 nucleotidesfor specific hybridization. Inhibitory polynucleotides include, withoutlimitation, antisense molecules, ribozymes, sense molecules andtriplex-forming molecules. In one embodiment, the inhibitorypolynucleotide is no more than about 50 nucleotides long.

While not wishing to be limited by theory, it is believed thatinhibitory polynucleotides inhibit the function of a target, in part, bybinding to the appropriate target sequence. An inhibitory polynucleotidecan inhibit DNA replication or DNA transcription by, for example,interfering with the attachment of DNA or RNA polymerase to the promoterby binding to a transcriptional initiation site or a template. It caninterfere with processing of mRNA, poly(A) addition to mRNA ortranslation of mRNA by, for example, binding to regions of the RNAtranscript such as the ribosome binding site. It can promote inhibitorymechanisms of the cells, such as promoting RNA degradation via RNaseaction. The inhibitory polynucleotide can bind to the major groove ofthe duplex DNA to form a triple helical or “triplex” structure. Methodsof inhibition using inhibitory polynucleotides therefore encompass anumber of different approaches to altering expression of specific genesthat operate by different mechanisms. These different types ofinhibitory polynucleotide technology are described in C. Helene and J.Toulme, (1990) Biochim. Biophys. Acta., 1049:99-125.

Antisense polynucleotides can include deoxyribonucleotides orribonucleotides. They can be chemically modified so as to improvestability in the body. Properties of the polynucleotide can beengineered to impart stability (e.g., nuclease resistance), tighterbinding or the desired T_(m). See, e.g., International patentpublication No. 94/12633.

The general approach to constructing various polynucleotides useful ininhibitory polynucleotide therapy has been reviewed by A. R. Vander Krolet al. (1988), Biotechniques 6:958-976, and by C. A. Stein et al.,(˜1988) Cancer Res. (1988) 48:2659-2668. See also Oligodeoxynucleotides:Antisense Inhibitors of Gene Expression, Cohen. J. S., editor. MacMillanPress. London pages 79-196 (1989), and Antisense RNA and DNA, (1988), D.A. Melton. Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.In certain embodiments inhibitory polynucleotides comprise a derivatizedsubstituent which is substantially non-interfering with respect tohybridization of the inhibitory polynucleotide to the targetpolynucleotide.

B. Antisense

This invention provides antisense polynucleotides capable ofspecifically hybridizing to a target sequence of Repro-PC-1.0. Antisensepolynucleotides are useful in vitro or in vivo to inhibit the activityof Repro-PC-1.0.

The antisense polynucleotides of this invention comprise an antisensesequence of at least 7 nucleotides that specifically hybridize to asequence from Repro-PC-1.0 and, more particularly, mammalianRepro-PC-1.0 and human Repro-PC-1.0.

The antisense sequence can be between about 10 and about 50 nucleotidesor between about 15 and about 35 nucleotides. In other embodiments,antisense polynucleotides are polynucleotides of less than about 100nucleotides or less than about 200 nucleotides. Accordingly, a sequenceof the antisense polynucleotide can specifically hybridize to all orpart of the Repro-PC-1.0, such as antisense polynucleotides to theRepro-PC-1.0 gene or its transcribed RNA. In one embodiment, thesequence of the polynucleotide contains within it the antisensesequence. In this case, the antisense sequence is contained within apolynucleotide of longer sequence. In another embodiment, the sequenceof the polynucleotide consists essentially of, or is, the antisensesequence. Thus, for example, the antisense polynucleotide can be apolynucleotide of less than about 50 nucleotides in a sequence thatspecifically hybridizes to the target sequence.

Generally, to assure specific hybridization, the antisense sequence issubstantially complementary to the target sequence in Repro-PC-1.0. Incertain embodiments, the antisense sequence is exactly complementary tothe target sequence. The antisense polynucleotides may includenucleotide substitutions, additions, deletions, or transpositions, solong as specific binding to the relevant target sequence correspondingto Repro-PC-1.0 mRNA or its gene is retained as a functional property ofthe polynucleotide.

The antisense polynucleotide should be long enough to form a stableduplex but short enough, depending on the mode of delivery, toadminister in vivo, if desired. The minimum length of a polynucleotiderequired for specific hybridization to a target sequence depends onseveral factors, such as G/C content, positioning of mismatched bases(if any), degree of uniqueness of the sequence as compared to thepopulation of target polynucleotides, and chemical nature of thepolynucleotide (e.g., methylphosphonate backbone, polyamide nucleicacid, phosphorothioate, etc.), among others.

For general methods relating to antisense polynucleotides, see AntisenseRNA and DNA, (1988), D. A. Melton, Ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.). For a review of antisense therapy, see, e.g.,Uhlmann et al., Chem. Reviews, 90:543-584 (1990).

C. Ribozymes

Cleavage of Repro-PC-1.0 can be induced by the use of ribozymes orcatalytic RNA. In this approach, the ribozyme would contain eithernaturally occurring RNA (ribozymes) or synthetic nucleic acids withcatalytic activity. Bratty et al., (1992) Biochim. Biophys. Acta.,1216:345-59 (1993) and Denhardt, (1992) Ann. N.Y. Acad. Sci., 660:70-76describe methods for making ribozymes.

Unlike the antisense and other polynucleotides described above, whichbind to an RNA or a DNA, a ribozyme not only binds but also specificallycleaves and thereby potentially inactivates a target RNA. Such aribozyme can comprise 5′- and 3′-terminal sequences complementary to theRepro-PC-1.0 RNA.

Optimum target sites for ribozyme-mediated inhibition of activity can bedetermined as described by Sullivan et al., PCT patent publication No.94/02595 and Draper et al., PCT patent publication No. 93/23569. Asdescribed by Hu et al., PCT patent publication No. 94/03596, antisenseand ribozyme functions can be combined in a single polynucleotide. Uponreview of the RNA sequence of Repro-PC-1.0 those in the art will notethat several useful ribozyme target sites are present and susceptible tocleavage by, for example, a hammerhead motif ribozyme.

Such engineered ribozymes can be expressed in cells or can betransferred by a variety of means (e.g., liposomes, immunoliposomes,biolistics, direct uptake into cells, etc.). Other forms of ribozymes(group I intron ribozymes (Cech (1995) Biotechnology 13: 323);hammerhead ribozymes (Edgington (1992) Biotechnology 10: 256) can beengineered on the basis of the disclosed Repro-PC-1.0 sequenceinformation to catalyze cleavage of Repro-PC-1.0 RNA. Moreover,ribozymes can comprise one or more modified nucleotides or modifiedlinkages between nucleotides, as described above.

D. Other Inhibitory Polynucleotides

In addition to the antisense and ribozyme inhibitory polynucleotides,one can construct polynucleotides that will bind to duplex nucleic acideither in the folded RNA component or in the gene for the RNA component,forming a triple helix-containing or triplex nucleic acid to inhibitRepro-PC-1.0 activity. Such polynucleotides of the invention areconstructed using the base-pairing rules of triple helix formation andthe nucleotide sequence of the RNA component (Cheng et al. (1988) J.Biol. Chem. 263: 15110; Ferrin and Camerini-Otero (1991) Science 354:1494; Ramdas et al. (1989) J. Biol. Chem. 264: 17395; Strobel et al.(1991) Science 254: 1639; Hsieh et al. (1990) op.cit.; Rigas et al.(1986) Proc. Natl. Acad. Sci. (U.S.A.) 83: 9591. Such polynucleotidescan block Repro-PC-1.0 activity in a number of ways, including bypreventing transcription of the Repro-PC-1.0 gene.

Typically, and depending on mode of action, the triplex-formingpolynucleotides of the invention comprise a sequence large enough toform a stable triple helix but small enough, depending on the mode ofdelivery, to administer in vivo.

E. Methods for Making Inhibitory Polynucleotides

Inhibitory polynucleotides can be made chemically or recombinantly.

1. Chemical Synthesis

Small inhibitory polynucleotides for direct delivery can be made bychemical synthesis. Chemically synthesized polynucleotides can be DNA orRNA, or can include nucleotide analogs or backbones that are not limitedto phosphodiester linkages.

2. Recombinant Production

For delivery into cells or for gene therapy methods, recombinantproduction of inhibitory polynucleotides through the use of expressionvectors is particularly useful. Accordingly, this invention alsoprovides expression vectors, e.g., recombinant polynucleotide moleculescomprising expression control sequences operatively linked to thenucleotide sequence encoding the inhibitory polynucleotide.

VII. Repro-PC-1.0 Polypeptides

This invention also provides purified, recombinant Repro-PC-1.0polypeptide and Repro-PC-1.0 analogs. Recombinant Repro-PC-1.0polypeptide includes the polypeptide whose amino acid sequence ispresented in SEQ ID NO:2, as well as allelic variants of it.Repro-PC-1.0 analogs include active Repro-PC-1.0 analogs, inactiveRepro-PC-1.0 analogs and immunogenic Repro-PC-1.0 analogs.

Repro-PC-1.0 polypeptide refers to native Repro-PC-1.0, the polypeptidewhose amino acid sequence is the amino acid sequence of SEQ ID NO:2, andto allelic variants of it. Polynucleotide molecules that encode allelicvariants of Repro-PC-1.0 are isolatable from prostate cancer cell cDNAor genomic DNA and typically hybridize under stringent conditions to thenucleotide sequence encoding Repro-PC-1.0 (SEQ ID NO:1). They can beobtained by amplification using, e.g., PCR primers taken from thesequence of Repro-PC-1.0 described herein.

Repro-PC-1.0 polypeptides are useful as immunogens to elicit theproduction of anti-Repro-PC-1.0 antibodies, as affinity capturemolecules to isolate such antibodies from a mixture, and as controls indiagnostic methods aimed at detecting Repro-PC-1.0 in a sample.

A Repro-PC-1.0 analog is a polypeptide whose sequence is not naturallyoccurring but is substantially identical over its sequence to a sequenceof native Repro-PC-1.0. Repro-PC-1.0 analogs include active Repro-PC-1.0analogs, inactive Repro-PC-1.0 analogs and immunogenic Repro-PC-1.0analogs. Repro-PC-1.0 analogs include fusion proteins, i.e.,polypeptides having a Repro-PC-1.0/Repro-PC-1.0 analog moiety fused withanother polypeptide moiety at its amino- or carboxy-terminal end.

Active Repro-PC-1.0 analogs have the biological activity ofRepro-PC-1.0. Repro-PC-1.0 is believed to function in exocytosis andendocytosis pathways. Generally, active Repro-PC-1.0 analogs have atleast 95% sequence identity with native Repro-PC-1.0. ActiveRepro-PC-1.0 analogs can be produced by, for example, introducingconservative amino acid substitutions into the sequence of nativeRepro-PC-1.0. Changes in the amino acid sequence outside the directrepeat sequences or outside amino acids 40-140 are most likely to betolerated. Amino acids around 1-15, 268-275 and 397-425 probably arehighly mutable. Amino acids in the transmembrane region, e.g., aminoacids 15-37 (SEQ ID NO:2) should not be substituted with hydrophilicamino acids. Active fragments of Repro-PC-1.0 can be identifiedempirically by truncating the protein from either the amino-terminus orthe carboxy-terminus to generate fragments, and testing the resultingfragments for Repro-PC-1.0 activity. More specifically, the followingsubstitutions in Repro-PC-1.0 are expected to yield analogs that haveRepro-PC-1.0 activity: N79S (i.e., substitute N at position 79 with S);V87L; S133A; E268D and V421M.) These substitutions occur in areas thatare not part of the following major motifs sharing identity with ratsynaptotagmin IV: PKA phosphorylation site, CK2 phosphorylation site,PKC phosphorylation site, N-myristoylation site, PKC C2 domain and thetransmembrane domain.

Inactive Repro-PC-1.0 analogs are polypeptides of at least 5 amino acidswhose amino acid sequence over its length is substantially identical tonative Repro-PC-1.0. Inactive Repro-PC-1.0 analogs include, for example,polypeptides encoding fragments of Repro-PC-1.0. Useful fragments ofRepro-PC-1.0 include polypeptides comprising the sequence of one or bothof the 116 amino acid direct repeats of Repro-PC-1.0. Another usefulfragment of Repro-PC-1.0 is one that lacks the transmembrane region and,therefore, does not anchor into the cell membrane. These inactiveanalogs are useful as inhibitory polypeptide mimics or decoys. Whenexpressed in a cell they compete with Repro-PC-1.0 polypeptides forinteraction with molecules that naturally interact with Repro-PC-1.0.Fragments generally are at least 10, 50 or 100 amino acids long.

Immunogenic Repro-PC-1.0 analogs are polypeptides having a sequence ofat least 5 amino acids selected from native Repro-PC-1.0 and which, whenpresented to an animal as an immunogen, elicit a humoral orcell-mediated immune response. This includes polypeptides comprising anamino acid sequence which is an epitope from Repro-PC-1.0, such asimmunogenic fragments of Repro-PC-1.0. Repro-PC-1.0 protein analogsoptionally are in isolated form.

Repro-PC-1.0 and analogs are most easily produced recombinantly, asdescribed herein. Recombinant Repro-PC-1.0 can be purified by affinitypurification. In one method, recombinant Repro-PC-1.0 analogs comprise apolyhistidine tag. The protein is purified on a nickel-chelate affinitymatrix. In another method. Repro-PC-1.0 is purified using an affinitymatrix carrying anti-Repro-PC-1.0 antibodies.

VIII. Antibodies and Hybridomas

In one aspect this invention provides a composition comprising anantibody that specifically binds Repro-PC-1.0 polypeptides. Antibodiespreferably have affinity of at least 10⁶ M⁻¹, 10⁷ M⁻¹, 10⁸ M⁻¹, or 10⁹M⁻¹. This invention contemplates both polyclonal and monoclonal antibodycompositions.

In one embodiment this invention provides immunotoxins againstRepro-PC-1.0-expressing cells. Immunotoxins are antibodies and the likeas described herein coupled to a compound, e.g., a toxin, that is toxicto a target cell. Toxins can include, for example, radioactive isotopes,ricin, cisplatin, antisense molecules, Diphteria toxin,Pseudomonasexotoxin A or Bacillus anthracis protective antigen.Immunotoxins bind to cancer cells that express Repro-PC-1.0 and killthem. They are useful in the therapeutic methods of this invention. Theantibodies of the invention have many uses. For example, such antibodiesare useful for detecting Repro-PC-1.0 polypeptides in immunoassays. Theantibodies also can be used to screen expression libraries forparticular expression products such as mammalian Repro-PC-1.0. These areuseful for detecting or diagnosing various pathological conditionsrelated to the presence of the respective antigens. Usually theantibodies in such a procedure are labeled with a moiety allowing easydetection of presence of antigen by antibody binding. Antibodies raisedagainst Repro-PC-1.0 can also be used to raise anti-idiotypicantibodies.

A. Production of Antibodies

A number of immunogens are used to produce antibodies that specificallybind Repro-PC-1.0 polypeptides. Full-length Repro-PC-1.0 is a suitableimmunogen. Typically, the immunogen of interest is a peptide of at leastabout 3 amino acids, more typically the peptide is 5 amino acids inlength, preferably, the fragment is 10 amino acids in length and morepreferably the fragment is 15 amino acids in length or greater. Thepeptides can be coupled to a carrier protein (e.g., as a fusionprotein), or are recombinantly expressed in an immunization vector.Antigenic determinants on peptides to which antibodies bind aretypically 3 to 10 amino acids in length. Naturally occurringpolypeptides are also used either in pure or impure form.

Recombinant polypeptides are expressed in eukaryotic or prokaryoticcells and purified using standard techniques. The polypeptide or asynthetic version thereof, is then injected into an animal capable ofproducing antibodies. Either monoclonal or polyclonal antibodies can begenerated for subsequent use in immunoassays to measure the presence andquantity of the polypeptide.

Methods for producing polyclonal antibodies are known to those of skillin the art. In brief, an immunogen preferably a purified polypeptide, apolypeptide coupled to an appropriate carrier (e.g., GST, keyhole limpethemanocyanin, etc.), or a polypeptide incorporated into an immunizationvector such as a recombinant vaccinia virus (see, U.S. Pat. No.4,722,848) is mixed with an adjuvant and animals are immunized with themixture. The animal's immune response to the immunogen preparation ismonitored by taking test bleeds and determining the titer of reactivityto the polypeptide of interest. When appropriately high titers ofantibody to the immunogen are obtained, blood is collected from theanimal and antisera are prepared. Further fractionation of the antiserato enrich for antibodies reactive to the polypeptide is performed wheredesired. See, e.g., Coligan (1991) Current Protocols in ImmunologyWiley/Greene, N.Y.; and Harlow and Lane (1989) Antibodies: A LaboratoryManual Cold Spring Harbor Press, N.Y.

Antibodies, including binding fragments and single chain recombinantversions thereof, against predetermined fragments of Repro-PC-1.0proteins are raised by immunizing animals, e.g., with conjugates of thefragments with carrier proteins as described above.

Monoclonal antibodies are prepared from cells secreting the desiredantibody. These antibodies are screened for binding to normal ormodified polypeptides, or screened for agonistic or antagonisticactivity, e.g., activity mediated through Repro-PC-1.0. In someinstances, it is desirable to prepare monoclonal antibodies from variousmammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies arefound in, e.g., Stites et al. (eds.) Basic and Clinical Immunology (4thed.) Lange Medical Publications, Los Altos, Calif., and references citedtherein; Harlow and Lane, Supra; Goding (1986) Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y.; andKohler and Milstein (1975) Nature 256: 495-497.

Other suitable techniques involve selection of libraries of recombinantantibodies in phage or similar vectors. See, Huse et al. (1989) Science246: 1275-1281: and Ward, et al. (1989) Nature 341: 544-546.

Frequently, the polypeptides and antibodies will be labeled by joining,either covalently or non-covalently, a substance which provides for adetectable signal. A wide variety of labels and conjugation techniquesare known and are reported extensively in both the scientific and patentliterature. Also, recombinant immunoglobulins may be produced. See,Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'lAcad. Sci. USA 86: 10029-10033.

The antibodies of this invention are also used for affinitychromatography in isolating Repro-PC-1.0 proteins. Columns are prepared,e.g., with the antibodies linked to a solid support, e.g., particles,such as agarose, Sephadex, or the like, where a cell lysate is passedthrough the column, washed, and treated with increasing concentrationsof a mild denaturant, whereby purified Repro-PC-1.0 polypeptides arereleased.

An alternative approach is the generation of humanized immunoglobulinsby linking the CDR regions of non-human antibodies to human constantregions by recombinant DNA techniques. See Queen et al., U.S. Pat. No.5,585,089.

A further approach for isolating DNA sequences which encode a humanmonoclonal antibody or a binding fragment thereof is by screening a DNAlibrary from human B cells according to the general protocol outlined byHuse et al., Science 246:1275-1281 (1989) and then cloning aidamplifying the sequences which encode the antibody (or binding fragment)of the desired specificity. The protocol described by Huse is renderedmore efficient in combination with phage display technology. See, e.g.,Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047. Phagedisplay technology can also be used to mutagenize CDR regions ofantibodies previously shown to have affinity for Repro-PC-1.0 proteinreceptors or their ligands. Antibodies having improved binding affinityare selected.

In another embodiment of the invention, fragments of antibodies againstRepro-PC-1.0 protein or protein analogs are provided. Typically, thesefragments exhibit specific binding to the Repro-PC-1.0 protein receptorsimilar to that of a complete immunoglobulin. Antibody fragments includeseparate heavy chains, light chains Fab, Fab′ F(ab′)₂ and Fv. Fragmentsare produced by recombinant DNA techniques, or by enzymic or chemicalseparation of intact immunoglobulins.

IX. Methods for Detecting Repro-PC-1.0 Polypeptides

Repro-PC-1.0 polypeptides can be identified by any methods known in theart. In one embodiment the methods involve detecting the polypeptidewith a ligand that specifically recognizes the polypeptide. Theantibodies of the invention are particularly useful for specificdetection of Repro-PC-1.0 polypeptides. A variety of antibody-baseddetection methods are known in the art. These include, for example,radioimmunoassay, sandwich immunoassays (including ELISA), Western blot,isolation on antibodies bound to a solid phase and in situ detectionwith labeled antibodies. Another method for detecting Repro-PC-1.0polypeptides involves identifying the polypeptide according to its massthrough, for example, gel electrophoresis, mass spectrometry or HPLC.Subject samples can be taken from any number of appropriate sources,such as blood, urine, tissue biopsy (e.g., lymph node tissue), etc.

X. Diagnostic, Monitoring and Prognostic Methods

Repro-PC-1.0 has been found in all prostate adenocarcinoma tissuetested. Also, it is virtually undetectable in cells other than prostatecancer cells. Therefore, Repro-PC-1.0 is both a highly selective andhighly specific marker for prostate cancer. Accordingly, the methodsdescribed herein for detecting Repro-PC-1.0 polynucleotides orRepro-PC-1.0 polypeptides in a sample are useful in methods fordiagnosing prostate cancer, monitoring its progress or treatment, anddetermining patient prognosis. The methods of the present inventionallow cancerous conditions to be detected with increased confidence andat an earlier stage, before cells are detected as cancerous based onpathological characteristics. It is, of course, understood bydiagnosticians that diagnostic tests are measured by their degree ofspecificity and sensitivity. Tests which are not perfectly specific orsensitive are, nevertheless, useful in diagnosis because they provideuseful information which, in combination with other evidence, canprovide a definitive diagnosis or indicate a course of treatment.

Methods for diagnosis involve determining a diagnostic amount ofRepro-PC-1.0 (e.g., mRNA, cDNA or polypeptide) in a patient sample andcomparing that amount with a normal range expected to be found in thesample. Repro-PC-1.0 mRNA or polypeptide has not been detected in normalprostate tissue, and the normal range is no higher than backgrounddetection. Therefore, any positive diagnostic amount detected abovebackground is a positive sign of prostate cancer. The samples used todetermine the normal range of Repro-PC-1.0 can be normal samples fromthe individual to be tested, or normal samples from other individualsnot suffering from the disease condition.

In one embodiment, methods of diagnosing prostate cancer involvedetecting Repro-PC-1.0 on the surface of prostate cancer cells. Cellsexpressing the protein display the amino terminal section ofRepro-PC-1.0 on their surfaces. Therefore, antibodies that recognize theamino terminus of Repro-PC-1.0 are particularly useful for this purpose.Such antibodies can be made, for example, by immunizing an animal with afragment of Repro-PC-1.0 that contains amino acids from 1 to about 15 ofSEQ ID NO:2. In this case, the test cells are exposed to a labeledantibody. Specific binding of the antibody to the cell indicates thatthe cell expresses Repro-PC-1.0 and is a positive indication that thecell is a prostate cancer cell.

Prostate cancer cells can metastasize even before the cancer isdetectable through histopathology. The diagnostic tests of thisinvention allow one to detect metastatic cells in, for example, thelymph nodes or the blood, by detecting Repro-PC-1.0.

A variety of patient samples can be used in the methods of theinvention. For example, cell extracts, cultured cells, or tissue samplesprovide convenient samples for use with the methods of the invention.The methods of the invention can use samples either in solution orextracts, for example, with RT-PCR, or samples such as tissue sectionsfor in situ methods of detection. Samples can also be obtained fromsources such as cells collected from bodily fluids and wastes, e.g.,urine, sputum, and blood; washes, e.g., bladder and lung; andfine-needle biopsies, e.g., from prostate; cellular materials; wholecells; tissue and cell extracts; RNA extracted from tissue and cells;and histological sections of tissue.

Methods for monitoring the course of prostate cancer involve determiningthe amount of Repro-PC-1.0 in a sample at a first and second time. Thetimes can be during routine physical examinations or during a course oftreatment for prostate cancer. As prostate cancer appears and/orprogresses, the amount of Repro-PC-1.0 in a sample is expected toincrease. Regression or cure of prostate cancer are accompanied by adecrease or elimination of Repro-PC-1.0 in a sample.

The stage of a rumor is an indication of patient prognosis. It has beenfound that more differentiated prostate cancer rumors, as determined byGleason staining, express higher levels of Repro-PC-1.0. Therefore, bycomparing a measured amount of Repro-PC-1.0 in a subject sample withamounts of Repro-PC-1.0 associated with various tumor stages, one canprovide a prognosis for the subject. Such a prognostic value is usefulin planning a course of treatment for the subject.

The diagnostic and prognostic methods can also be carried out inconjunction with other diagnostic or prognostic tests. In someinstances, such combination tests can provide useful informationregarding the progression of a disease, although the present methods fortesting for Repro-PC-1.0 provide much useful information in this regard.

Another diagnostic method of the invention involves the administrationto a subject of a labeled composition that specifically binds to cellsbearing Repro-PC-1.0, such as labelled antibodies. Then, thelocalization of the label is determined by any of the known radiologicmethods. Any conventional method for visualizing diagnostic imaging canbe used. Usually gamma- and positron-emitting radioisotopes are used forcamera imaging and paramagnetic isotopes are used for MRI.

The Repro-PC-1.0 gene is located on chromosome 18. A translocation atthis site can result in alteration of Repro-PC-1.0 activity, such asactivated transcription or changed function. Chromosomal translocationsin the vicinity of the Repro-PC-1.0 gene can be detected by hybridizinga labeled probe of this invention to a chromosome spread. Atranslocation, duplication or deletion can be identified by aberranthybridization patterns compared to normal. Such tests are useful indetecting genetic abnormalities such as familial disposition to prostatecancer, or early onset of the disease. A method for fluorescent in situhybridization of chromosomes is provided in the Examples.

The present invention also provides for kits for performing thediagnostic and prognostic method of the invention. Such kits include apolynucleotide probe or primer, or an antibody specific for Repro-PC-1.0and instructions to use the reagents to detect Repro-PC-1.0 in a patientsample.

XI. Methods for Inhibiting Repro-PC-1.0 Expression or Activity and ofTreating Prostate Cancer

Inhibiting Repro-PC-1.0 expression or activity changes a prostate cancercell from metastatic to non-metastatic. Inhibiting Repro-PC-1.0expression or activity is useful in vitro in the prophylactic andtherapeutic treatment or prostate cancer or other conditions involvingRepro-PC-1.0 expression. Accordingly, this invention provides methodsfor inhibiting Repro-PC-1.0 expression or activity. The methods involvecontacting a prostate cancer cell, in vitro or in vivo, with aninhibitory polynucleotide, an immunotoxin or another compound thatinhibits Repro-PC-1.0 expression or activity.

A. Delivery of Inhibitory Polynucleotides

This invention contemplates a variety of means for delivering aninhibitory polynucleotide to a subject including, for example, directuptake of the molecule by a cell from solution, facilitated uptakethrough lipofection (e.g., liposomes or immunoliposomes),particle-mediated transfection, and intracellular expression from anexpression cassette having an expression control sequence operablylinked to a nucleotide sequence that encodes the inhibitorypolynucleotide. Methods useful for delivery of polynucleotides fortherapeutic for therapeutic purposes are described in Inouye et al.,U.S. Pat. No. 5,272,065.

B. Pharmaceutical Compositions and Treatment

Agents, such as inhibitory polynucleotides, immunotoxins or othercompounds that inhibit Repro-PC-1.0 expression or activity preferablyare delivered in pharmaceutical compositions comprising the agent and apharmaceutically acceptable carrier. The agent can be administered byany route that gives it access to cells expressing Repro-PC-1.0, forexample, prostate rumor cells. This includes, for example, aqueoussolutions for enteral, parenteral or transmucosal administration, e.g.,for intravenous administration, as tonics and administration to mucousor other membranes as, for example, nose or eye drops; solid and othernon-aqueous compositions for enteral or transdermal delivery, e.g., aspills, tablets, powders or capsules; transdermal or transmucosaldelivery systems for topical administration, and aerosols or mists fordelivery by inhalation. One advantage of delivery by a mode that is easyto administer, e.g., enteral or by intravenous or intramuscularinjection is that such modes mimic possible modes of delivery should theagent be formulated as a pharmaceutical.

In one embodiment, the pharmaceutical composition is in the form of aunit dose which contains a pharmacologically effective amount of theRepro-PC-1.0-inhibitory compound. The unit dose, taken as part of atherapeutic regimen, results in inhibition of growth of prostate cancercells. Thus, the pharmaceutical compositions of the invention, whateverthe form, are administered in a pharmacologically effective amount tothe subject.

The amount of the pharmaceutical composition delivered, the mode ofadministration and the time course of treatment are at the discretion ofthe treating physician. Prophylactic treatments are indicated forpersons at higher than average risk of getting prostate cancer, forexample, persons with elevated PSA, PAP (prostate acid phosphatase) orPSP (prostate specific protein) levels. Therapeutic treatments areindicated for persons diagnosed with prostate cancer.

XII. Hormone Therapy

We have shown that prostate rumors grown in males become malignant,while those grown in females remain benign. While not wishing to belimited by theory, it is believed that this difference is due to thehormonal environment. It is believed that up-regulation of Repro-PC-1.0expression is directly or indirectly controlled by a hormone responseelement that is responsive to androgens, such as testosterone, but notto estrogens. However, regardless of the exact role Repro-PC-1.0expression plays in prostate cancer, the experiments reported hereinshow that altering the hormonal environment changes the character ofprostate cells.

Therefore, one method for treating prostate cancer involves inhibitingcontact between the prostate cancer cell and a hormone that activateshormone response elements regulating the malignant condition. In oneembodiment, prostate cancer is treated by decrease the amount oftestosterone in the system of the subject, or increasing the amount ofestrogen. Methods for altering the hormone profile of subjects is wellknown in the art. For example, estrogens can be administered to increasetheir amount in the blood, and compounds, such as flutamide, luteinizinghormone releasing hormone (LHRH) antagonists and cyproterone acetate areknown to decrease the amount of androgens in the system.

XIII. Polypeptide and Polynucleotide Vaccines Against Repro-PC-1.0

This invention also provides vaccines against Repro-PC-1.0-expressingcells and methods for using them.

In one aspect, this invention provides methods for eliciting a humoralimmune response against Repro-PC-1.0. The method involves immunizing asubject with a vaccine comprising an immunogenic amount of Repro-PC-1.0polypeptide or an immunogenic Repro-PC-1.0 analog. Such vaccines elicitantibodies against Repro-PC-1.0.

In another aspect, this invention provides methods for eliciting an MHCClass II-dependent immune response against cells expressingRepro-PC-1.0. MHC Class II molecules bind peptides having particularamino acid motifs well known in the art. The MHC Class II-dependentresponse involves the uptake of an antigen by antigen-presenting cells(APC's), its processing, and presentation on the cell surface as part ofan MHC Class II/antigenic peptide complex. Alternatively, MHC Class IImolecules on the cell surface can bind peptides having the proper motif.

Antigen presenting cells interact with CD4-positive T-helper cells,thereby activating the T-helper cells. Activated T-helper cellsstimulate B-lymphocytes to produce antibodies against the antigen.Antibodies mark cells bearing the antigen on their surface. The markedcells are subject to antibody-dependent cell-mediated cytotoxicity, inwhich NK cells or macrophages, which bear Fc receptors, attack themarked cells.

Methods for eliciting an MHC Class I-dependent immune response involveadministering to a subject a vaccine including an immunogenic amount ofRepro-PC-1.0 polypeptide or an immunogenic Repro-PC-1.0 analog thatincludes an amino acid motif recognized by MHC Class II molecules of thesubject. Alternatively, antigen presenting cells can be cultured withsuch peptides to allow binding, and the cells can be administered to thesubject. Preferably, the cells are syngeneic with the subject.

In another aspect, this invention provides methods for eliciting an MHCClass I-dependent cell-mediated immune response against cells expressingRepro-PC-1.0 in a subject. MHC Class I molecules also bind peptideshaving particular amino acid motifs well known in the art. Proteinsexpressed in a cell are digested into peptides become associated withMHC Class I molecules and are presented on the cell surface. There, theyare recognized by CD8-positive lymphocytes, generating a cytotoxicT-lymphocyte response against cells expressing the epitopes inassociation with MHC Class I molecules. Because prostate cancer cellsexpress Repro-PC-1.0, the generation of cytotoxic T-lymphocytes thatattack such cells is useful in the prophylactic or therapeutic treatmentof prostate cancer.

HLA-A1 binding motif includes a first conserved residue of T, S or M, asecond conserved residue of D or E, and a third conserved residue of Y.Other second conserved residues are A, S or T. The first and secondconserved residues are adjacent and are preferably separated from thethird conserved residue by 6 to 7 residues (SEQ ID NOS:11 and 12). Asecond motif consists of a first conserved residue of E or D and asecond conserved residue of Y where the first and second conservedresidues are separated by 5 to 6 residues (SEQ ID NOS:13 and 14). TheHLA-A3.2 binding motif includes a first conserved residue of L, M, I, V,S, A, T and F at position 2 and a second conserved residue of K, R or Yat the C-terminal end. Other first conserved residues are C, G or D andalternatively E. Other second conserved residues are H or F. The firstand second conserved residues are preferably separated by 6 to 7residues (SEQ ID NOS:15 and 16). The HLA-A11 binding motif includes afirst conserved residue of T or V at position 2 and a C-terminalconserved residue of K. The first and second conserved residues arepreferably separated by 6 to 7 residues (SEQ ID NOS:17 and 18). TheHLA-A24.1 binding motif includes a first conserved residue of Y, F or Wat position 2 and a C terminal conserved residue of F, I, W, M or L. Thefirst and second conserved residues are preferably separated by 6 to 7residues (SEQ ID NOS:19 and 20).

A methodical way of identifying peptides that can activate a cytotoxicT-cell response is to prepare a set of overlapping fragments of thepolypeptide of about 9 to 15 amino acids, and test them for the abilityto stimulate T-cells. For example, the set could include fragmentsspanning amino acids 1-15, 2-16, 3-17, etc. Such fragments can beroutinely prepared by a peptide synthesizer and can be tested in batchesto decrease the amount of experimentation necessary. For example, theset of 25 overlapping fragments including fragment 1-15 to fragment25-39 can be tested as compared to a set including fragment 26-40 to51-65. If only one set shows activity, that set can be broken down intotwo smaller sets and tested again, until individual immunogenic peptidesare identified.

Another method involves transfecting cells ex vivo with such expressionvectors, and administering the cells to the subject. The cellspreferably are syngeneic to the subject.

Methods for eliciting an immune response against Repro-PC-1.0 in asubject are useful in prophylactic methods for preventing prostatecancer when the vaccine is administered to a subject who does notalready suffer from prostate cancer.

This invention also provides a method for activating cytotoxicT-lymphocytes against cells that express a Repro-PC-1.0 comprisingcontacting T-lymphocytes ex vivo with an immunogenic peptide comprisinga linear epitope derived from the Repro-PC-1.0, the peptide capable ofinducing a Class I MHC-restricted cytotoxic T-lymphocyte responseagainst cells expressing the Repro-PC-1.0.

This invention also provides a method for determining whether a cellexpresses Repro-PC-1.0 polypeptide involving the steps of contacting thecell with a cytotoxic T-lymphocyte activated against cells expressing aRepro-PC-1.0 and determining whether the cytotoxic T-lymphocyte attacksthe cell.

This invention also provides an immunogenic peptide comprising a linearepitope derived from the Repro-PC-1.0, the peptide capable of inducing aClass I MHC-restricted cytotoxic T-lymphocyte response against cellsexpressing Repro-PC-1.0. In certain embodiments, the immunogenic peptidehas between 8 and 12 amino acids and the linear epitope has a Class IMHC molecule binding motif. In another embodiment, the immunogenicpeptide further comprises a T helper cell epitope.

The following chart provides portions of the amino acid sequence ofRepro-PC-1.0 (SEQ ID NO:2). Amino acid numbers are indicated. Bracketedbars over the amino acid sequence indicate vertebrate MHC Class I or MHCClass II binding motifs. Amino acid numbers are indicated. Peptides ofabout 8-15 amino acids in length that include these motifs, includingpeptides whose entire amino acid sequence is selected from the sequenceof Repro-PC-1.0, bind to MHC molecules and can be used to induce acell-mediated or humoral immune response against Repro-PC-1.0.

          |--|   |---| (SEQ ID NO:21) 9 eefdeiptvvgifsafglvftvslfawiccq          19 22 25  29              |--| (SEQ ID NO:22)          |--|      |---| 42 ssksnktppykfvhvlkgvdiypenlnskkkfga          52 55    61  65              55 58           |---| (SEQ IDNO:23) 114 spsdlenatpklflegekesvspes           124 128              |--| (SEQ ID NO:24)          |--|         |--|            |---|             |--| 199lpekkhkvktrvlrktldpafdetftfygipytqiqelalhftilsfdrfsrddiigevl          209         221           235 239          252              213            |---|  |--|            |---| (SEQ ID NO:25)259 iplsgielsegkmlmnreiikrnvrkssgrgellislcyqsttntl            270274  279        290 294           |--|     |---| (SEQ ID NO:26) 376vldsergsrnevigqlvlgaaaegtggehwkei           386     394 398

This invention also provides a pharmaceutical composition capable ofeliciting an immune response against Repro-PC-1.0 (e.g., a vaccine)comprising a pharmaceutically acceptable carrier and an immunogenicamount of a compound selected from Repro-PC-1.0, a fragment ofRepro-PC-1.0, an immunogenic peptide comprising a linear epitope derivedfrom Repro-PC-1.0, the peptide capable of inducing a Class IMHC-restricted cytotoxic T-lymphocyte response or a Class IIMHC-restricted immune response against cells expressing Repro-PC-1.0.

XIV. Transgenic Non-Human Animals

This invention also provides non-human mammals transgenic forRepro-PC-1.0. As used herein, “animal transgenic for Repro-PC-1.0”refers to an animal, in particular a mammal, whose germ cells (i.e.,oocytes or sperm), at least, comprise a recombinant nucleic acidmolecule comprising expression control sequences operatively linked to anucleic acid sequence encoding Repro-PC-1.0. Such animals are useful,for example, as models in the study of prostate cancer.

In one embodiment, the expression control sequences are not naturallyfound operatively linked to Repro-PC-1.0. In one embodiment, therecombinant nucleic acid comprises a non-native Repro-PC-1.0 codingsequence, i.e., a Repro-PC-1.0 sequence that the species does notproduce in nature. In one embodiment, the Repro-PC-1.0 is a humanRepro-PC-1.0. In another embodiment, the expression control sequencesare non-native expression control sequences introduced into the germcells so as to recombine with the naturally occurring gene and controlits expression. Particularly useful transgenic mammals of this inventioninclude rabbits and rodents such as mice.

The transgenic animals of this invention are produced, for example, byintroducing the recombinant nucleic acid molecule into a fertilized eggor embryonic stem (ES) cell, typically by microinjection,electroporation, lipofection, particle-mediated gene transfer. Thetransgenic animals express the heterologous nucleotide sequence intissues depending upon whether the promoter is inducible by a signal tothe cell, or is constitutive. Transgenic animals can be bred withnon-transgenic animals to produce transgenic animals with mixedcharacteristics.

XV. Methods for Screening for Compounds that Regulate Expression orActivity of Repro-PC-1.0

Compounds that regulate the expression or activity of Repro-PC-1.0 arecandidates as therapeutic agents in the treatment of prostate cancer.This invention provides methods for determining whether a compoundregulates (e.g., activates or inhibits) expression or activity ofRepro-PC-1.0.

Methods for determining whether a compound regulates Repro-PC-1.0expression involve administering to a cell or a test animal having anexpressible Repro-PC-1.0 gene with the compound, and determining whetherexpression Repro-PC-1.0 is altered. In one embodiment, the methodsinvolve administering the compound to a culture comprising the cell orto a test animal that has cells expressing Repro-PC-1.0, measuring theamount of the Repro-PC-1.0 polynucleotide or polypeptide in a samplefrom the culture or the animal, and determining whether the measuredamount is different than the amount in a sample from the culture or fromthe animal under control conditions (e.g., to which no compound has beenadministered). Statistically significant (p<0.05) differences betweenthe amount measured from the test sample and from the control sample arerecorded and indicate that the compound alters the amount ofRepro-PC-1.0 produced by the cell.

Methods for determining whether a compound regulates the biologicalactivity of Repro-PC-1.0 involve contacting a cell that expressesRepro-PC-1.0 with the compound and determining whether Repro-PC-1.0activity is altered. Repro-PC-1.0 has a significant level of amino acidsequence identity with the synaptotagmins: therefore it is believed thatRepro-PC-1.0 functions in the membrane fusion and membrane buddingreactions in exocytosis. Exocytosis is the process by which anintracellular vesicle fuses with the plasmalemma. As a result, thevesicle contents are released into the extracellular medium (secretion)and the components of the vesicle membrane become part of theplasmalemma. Some vesicles fuse with the plasmalemma constitutively,whereas others accumulate under the plasmalemma and fuse only after anappropriate stimulus. Constitutive exocytosis is used by cells fornon-regulated protein secretion and for insertion of newly synthesizedprotein components into the plasmalemma. Regulated exocytosis ischaracteristic of secretory granules derived from the Golgi complex,which store concentrated secretory products, and of specialized vesiclessuch as synaptic vesicles, which store neurotransmitters.

Activity assays include detecting changes in the levels of secretionsinto the media or changes in membrane capacitance after the introductionof potential inhibitors of Repro-PC-1.0 such as anti-sense nucleotides,anti-Repro-PC-1.0 antibodies, peptide fragments of Repro-PC-1.0 or othermolecules in combinatorial libraries. Immunological methods are used fordetecting changes in the levels of released molecules such as, but notlimited to PSA, PAP, PSP into the media, with, e.g., commerciallyavailable test kits (J. Steroid Biochem. Molec. Biol., 37:849 (1990)).Neurotransmitter or ATP release can be detected by HPLC or byluciferin-luciferase assays, respectively (Science, 256:1820 (1992)).Alternatively, cells can be radiolabelled and the release of secretorygranule contents could be measured by scintigraphy (Cell, 70:765(1992)). Increased exocytosis can also be detected by monitoringincreases in membrane capacitance. In this case, transmembrane currentis measured using conventional whole-cell patch-clamp methods (Neuron,10:21 (1993); Nature, 364:540 (1993)).

The compound to be tested can be selected from a number of sources. Forexample, combinatorial libraries of molecules are available forscreening experiments. Using such libraries, thousands of molecules canbe screened for regulatory activity. In one preferred embodiment, highthroughput screening methods involve providing a library containing alarge number of potential therapeutic compounds (candidate compounds).Such “combinatorial chemical libraries” are then screened in one or moreassays, as described herein, to identify those library members(particular chemical species or subclasses) that display a desiredcharacteristic activity. The compounds thus identified can serve asconventional “lead compounds” or can themselves be used as potential oractual therapeutics.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res. 37:487-493, Houghton et al. (1991) Nature, 354: 84-88). Peptide synthesisis by no means the only approach envisioned and intended for use withthe present invention. Other chemistries for generating chemicaldiversity libraries can also be used. Such chemistries include, but arenot limited to: peptoids (PCT Publication No WO 91/19735, 26 December1991), encoded peptides (PCT Publication WO 93/20242, 14 Oct. 1993),random bio-oligomers (PCT Publication WO 92/00091, 9 Jan. 1992),benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993) Proc.Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara etal. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimeticswith a Beta-D-Glucose scaffolding (Hirschmann et al., (1992) J. Amer.Chem. Soc. 114: 9217-9218), analogous organic syntheses of smallcompound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 116: 2661),oligocarbamates (Cho, et al., (1993) Science 261:1303), and/or peptidylphosphonates (Campbell et al., (1994) J. Org. Chem. 59: 658). See,generally, Gordon et al., (1994) J. Med. Chem. 37:1385, nucleic acidlibraries, peptide nucleic acid libraries (see, e.g., U.S. Pat. No.5,539,083) antibody libraries (see, e.g., Vaughn et al. (1996) NatureBiotechnology, 14(3): 309-314), and PCT/US96/10287), carbohydratelibraries (see, e.g., Liang et al. (1996) Science, 274: 1520-1522, andU.S. Pat. No. 5,593,853), and small organic molecule libraries (see,e.g., benzodiazepines, Baum (1993) C&EN, Jan 18, page 33, isoprenoidsU.S. Pat. No. 5,569,588, thiazolidinones and metathiazanones U.S. Pat.No. 5,549,974, pyrrolidines U.S. Pat. Nos. 5,525,735 and 5,519,134,morpholino compounds U.S. Pat. No. 5,506,337, benzodiazepines 5,288,514,and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.).

In one embodiment this invention provides inhibitory compounds thatinhibit expression of Repro-PC-1.0 identified or identifiable by thescreening methods of this invention.

XVI. Genomics

The identification of cognate or polymorphic forms of the Repro-PC-1.0gene and the tracking of those polymorphisms in individuals and familiesis important in genetic screening. Accordingly, this invention providesmethods useful in detecting polymorphic forms of the Repro-PC-1.0 gene.The methods involve comparing the identity of a nucleotide or amino acidat a selected position from the sequence of a test Repro-PC-1.0 genewith the nucleotide or amino acid at the corresponding position from thesequence of native Repro-PC-1.0 (SEQ ID NO:1). The comparison can becarried out by any methods known in the art, including direct sequencecomparison by nucleotide sequencing, sequence comparison ordetermination by hybridization or identification of RFLPs.

In one embodiment, the method involves nucleotide or amino acidsequencing of the entire test polynucleotide or polypeptide, or asubsequence from it, and comparing that sequence with the sequence ofnative Repro-PC-1.0. In another embodiment, the method involvesidentifying restriction fragments produced upon restriction enzymedigestion of the test polynucleotide and comparing those fragments withfragments produced by restriction enzyme digestion of nativeRepro-PC-1.0 gene. Restriction fragments from the native gene can beidentified by analysis of the sequence to identify restriction sites.Another embodiment involves the use of oligonucleotide arrays. (See,e.g., Fodor et al., U.S. Pat. No. 5,445,934.) The method involvesproviding an oligonucleotide array comprising a set of oligonucleotideprobes that define sequences selected from the native Repro-PC-1.0sequence, generating hybridization data by performing a hybridizationreaction between the target polynucleotide molecules and the probes inthe set and detecting hybridization between the target molecules andeach of the probes in the set and processing the hybridization data todetermine nucleotide positions at which the identity of the targetmolecule differs from that of native Repro-PC-1.0. The comparison can bedone manually, but is more conveniently done by a programmable, digitalcomputer.

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLES

I. LNCaP Tumor Xenografts Display Morphological and MetastaticCharacteristics that are Dependent Upon the Environment in Which Theyare Generated

Using a model system of LNCaP human prostate rumor cells, we raisedtumors in both male and female athymic mice. The tumors, which typicallytook at least 2-3 months to develop into palpable masses, showedremarkably different features depending on whether they were propagatedin a male or a female environment. Tumors that developed in male mice(male-LNCaP) showed extensive vascularization and morphologicaldestruction compared to the highly regular and homogeneous compositionof the tumors raised in females (female-LNCaP) (FIG. 1). Additionally,the tumors raised in male hosts had gained metastatic potential, whereasno metastases were ever detected in female mice. The differentialmorphologies of these tumors suggested the possibility that specificsequences involved in metastatic progression and/or angiogenesis hadbeen induced in the male environment.

The aberrant expression of a number of oncogenes and growth factors(up-regulation) as well as tumor suppressor genes (down-regulation) hasbeen implicated in a number of human tumors and in cancer progression ingeneral. We therefore examined the expression of a representative panelof these sequences in order to investigate whether the differentialappearance of male derived LNCaP tumors was the result of an androgendriven up- or down-regulation of a previously characterized factor. RNAisolated from male-LNCaPs, female-LNCaPs, LNCaP cells, PC-3 cells (anon-androgen responsive human prostate cell line), normal human spleentissue and normal human liver tissue was subjected to Northernhybridization using a variety of probes for previously characterizedoncogenes, growth factors and tumor suppressors. As can be seen in Table1, there was no difference in the pattern of expression detected inmale-LNCaP tumors compared to female-LNCaP tumors or to LNCAP cellsgrown in vitro indicating that the differentiated state of male-LNCaPtumors was not due to a differential expression of any of these morecommon factors.

TABLE 1 ONCOGENE EXPRESSION TO LNCaP TUMORS Summary of Northernhybridization results for a variety of oncogene probes to 4 μg total RNAfrom different tissues, cell lines and tissues. ^(a-f), The presence ofdetectable RNA was scored on a relative basis. +, detectable levels; −,undetectable levels. LNCaP^(a) LNCaP^(b) Probe (Male) (Female) LNCaP^(c)PC-3^(d) Spleen^(e) Liver^(f) FGF, acidic + + + + − − FGF, basic + + + +− − PDGF, A-chain + + + + − + PDGF, B-chain + + + + + +PDGF-receptor + + + + − − TGF-alpha + + + + − − TGF-B + + + + − −c-myc + + + + − − EGF-receptor + + + + − − Ha-ras + + + + + +Ki-ras + + + + − − NM23 + + + + − − c-myc (oligo) + + + − − − p53(oligo) + + + + + + androgen + + + + − − receptor (oligo)TNF-alpha + + + + − + (oligo) TNF-B (oligo) + + + + − − int-2(oligo) + + + + + + actin + + + + + + alpha-tubulin + + + + + + ^(a)RNAisolated from LNCaP prostate adenocarcinoma tumors propagated in maleathymic mice. ^(b)RNA isolated from LNCaP prostate adenocarcinoma tumorspropagated in female athymic mice. ^(c)RNA isolated from LNCaP prostateadenocarcinoma cells. ^(d)RNA isolated from PC-3 (non-androgenresponsive) cells. ^(e)RNA isolated from human spleen tissue. ^(f)RNAisolated from human liver tissue.

II. Isolation of Male-LNCaP-Specific Sequences

In order to isolate sequences that are over-expressed in male-LNCaPtumors and that might elucidate the mechanism(s) responsible for thedramatic morphological differences, we generated a male-LNCaP-specificprobe by three rounds of subtractive hybridization with female-LNCaPtumor cDNA. This male-LNCaP specific probe was then used to perform aprimary screen of a lambda-ZAP-male-LNCaP tumor cDNA library. Positiveplaques were subjected to a dual secondary screen, using the male-LNCaPspecific probe and total female-LNCaP tumor cDNA. Clones were consideredpositive if they hybridized strongly to the “male-specific” probe andweakly to the female probe.

The resulting positives were subjected to a tertiary screen in which theclones were “rescued” and their plasmid DNA was subjected to duplicateSouthern hybridizations using total male-LNCaP or total female-LNCaPcDNA. The DNA from clones hybridizing more strongly to male-LNCaPsequences was then subjected to Northern analyses. The DNA from oneclone, Repro-PC-1.0, when hybridized to equivalent amounts of RNA frommale-LNCaP tumors, female-LNCaP tumors or LNCAP cells showed an 10×amplification of a single 4.4 kb mRNA in male-LNCaP tumors (FIG. 2 a).Rehybridization of the same blot with probes for actin and tubulinshowed that the amplified signal detected by Repro-PC-1.0 in male-LNCaPtumors was not due to an increased level of RNA in that lane (FIG. 2 b).

III. Sequence Analysis of Repro-PC-1.0

Initial sequence analysis of clone Repro-PC-1.0 did not reveal anysignificant open reading frames (ORFs) in either direction.Subsequently, an overlapping clone (PS5-1), was isolated from themale-LNCaP tumor library by hybridization with an oligonucleotide probeencoding 5′ sequences contained within the Repro-PC-1.0 insert.Directionality of the Repro-PC-1.0 clone was inferred from the presenceof a putative poly-A tail. The complete coding region was determined bysequencing PS5-1 and an overlapping RACE-PCR derived 5′ end cDNA clone.The alignment of these clones is shown in FIG. 3. Northern analyses ofthe 2 overlapping clones to panels of RNA isolated from male-LNCaPtumors, female-LNCaP tumors, LNCaP cells and PC-3 cells revealed thesame pattern of hybridization as originally observed with Repro-PC-1.0.

Sequence analysis of the overlapping clones revealed a single 1275 bpORF encoding 425 amino acids, followed by a 2466 bp 3′ untranslatedregion to which a polyadenylate tail was added (SEQ ID NO: 1). Thesequence obtained for the 5′ untranslated region of Repro-PC-1.0 wasmuch shorter than the approximately 750 bp of sequence predicted, basedon the size of the 4.4 kb message that was observed by RNA blothybridizations, and is therefore probably incomplete. The M_(r) and pIwere calculated to be 48,070 and 8.83, respectively.

Sequence identity and similarity were determined using the FASTDBprogram, Bionet suite, Oxford Molecular Group, Campbell, Calif. orBLASTX, NCBI.

The predicted amino acid sequence of the C-terminal region ofRepro-PC-1.0 contained two copies of a 116 amino acid direct repeat thathad 34% identity (41% similarity) with each other. These repeats arelocated from amino acid 150 to 252 and amino acid 276 to 396. Therepeats were found to be homologous to the C2 regulatory domain ofcalcium-dependent isoforms of protein kinase C (PKC), and to isoforms ofsynaptotagmin (FIG. 4).

Synaptotagmin is actually a family of highly conserved, abundantsynaptic vesicle proteins that has been proposed to play a role insynaptic vesicle translocation to the presynaptic release site of theplasma membrane (docking) and/or fusion of these two membranes.Structurally, synaptotagmin isoforms can be divided into several domainswhich include: an intravesicular, amino-terminal domain; a singletransmembrane domain; and a cytoplasmic, carboxyl-terminal domain thatconsists of two repeats homologous to the C2 regulatory domain of PKCtermed A and B (FIG. 4). Homologues of synaptotagmin have shown greaterconservation of sequence identity and similarity in the cytoplasmicdomain containing the two PKC C2-homologous repeats than in theN-terminal intravesicular or transmembrane domains.

FIG. 5 shows the alignment of the amino acid sequences for Repro-PC-1.0and rat synaptotagmin IV. Repro-PC-1.0 shows 90% overall identity withrat synaptotagmin IV. Like the other synaptotagmin isoforms,Repro-PC-1.0 was most similar to these sequences in the PKC C2 repeatC-terminal region (91% identity). The two internal repeats ofRepro-PC-1.0 are approximately as homologous to each other (34%identity) as to the corresponding region of PKC (identity between 35%and 43# depending on the isoform). As in the other forms ofsynaptotagin, the amino acid residues that are identical between the twointernal repeats of Repro-PC-1.0 are also conserved between Repro-PC-1.0and PKC, revealing a core consensus sequence of SDPY(V/I)K (SEQ IDNO:27) followed by a stretch of basic residues (FIG. 6).

Hydrophobicity plots of the amino acid sequence of Repro-PC-1.0 revealeda single segment, from residues 15-37, of sufficient length andhydrophobicity to constitute a transmembrane domain. Although thisdomain does not align colinearly with the corresponding domain-in theother synaptotagmins, it also displays the unusual transmembraneboundaries reported for other synaptotagmins. The N-terminal border ofthe putative transmembrane domain is flanked by a proline, theC-terminus of the domain is flanked by cysteine residues followed by ahighly positively charged region.

IV. Chromosomal Location

Southern blot analyses of Repro-PC-1.0 hybridization to human genomicDNA revealed a non-complex pattern indicating a single copy sequence.Analysis of Repro-PC-1.0 hybridization to genomic DNAs from a panel ofphylogenetically distinct species showed that the sequences encodingRepro-PC-1.0 were highly conserved, hybridizing to yeast DNA even underhigh stringency conditions. A single, evolutionarily conservedRepro-PC-1.0 gene localizes to chromosome 18.

V. Expression of Repro-PC-1.0 in Prostate Carcinoma and in Other Tissues

In order to compare the expression of Repro-PC-1.0 in normal prostate toprostate adenocarcinomas and benign hyperplasias, Repro-PC-1.0 sequenceswere specifically amplified from RNA isolated from a number of wellcharacterized tissue sources by RT-PCR. These products werefractionated, transferred and hybridized with a Repro-PC-1.0 probe andthe level of expression was graded by relative signal intensity of theRepro-PC-1.0 specific bands (Table 2).

TABLE 2 EXPRESSION OF REPRO-PC-1.0 IN PROSTATE CARCINOMA The level ofexpression was graded by relative signal intensity of the Repro-PC-1.0specific bands: Repro-PC-1.0 PCR Tissue Source Hybridization LNCaP Tumor(male)* +++ + DNase +++ + RNase − Normal Prostate** − + DNase − + RNase− Prostate Adenocarcinoma*** ++ + DNase + + RNase − Prostatic BenignHyperplasia − Uterus − +++ very strong hybridization ++ stronghybridization + moderate hybridization − no hybridization *LNCaP Tumortissue grown in male athymic mice. **The prostate was obtained from a 65yr. old male, who may have had the beginning of undiagnosed prostatecancer. ***Compilation of analysis from 2 different prostateadenocarcinomas.

Repro-PC-1.0 sequences were only present in the RNAs isolated fromprostatic adenocarcinoma samples and were not detectable in samplesrepresenting benign hyperplasias. The marginal signal detected in normalprostate tissue may reflect the beginning of undiagnosed prostatecancer, as the source of the tissue was an elderly man. To ensure thatthe amplified sequences were not due to genomic DNA contaminants,control reactions were performed, adding RNase or DNase prior to the 1ststrand synthesis step. As shown in Table 2, the addition of DNase had noeffect of the level of signal detected in prostate adenocarcinomasamples, whereas RNase treatment eradicated the signal, indicating thatthe Repro-PC-1.0 hybridizing signal detected in the adenocarcinomassamples was due to Repro-PC-1.0 RNA sequences and not due tocontaminating genomic sequences.

TABLE 3 REPRO-PC-1.0 mRNA IN HUMAN CELL LINES AND MAMMALIAN TISSUESSummary of Northern hybridization results for different human cell linesand mammalian tissues. CELL LINE EXPRESSION TISSUE SOURCE Repro-PC-1.0LNCaP prostate adenocarcinoma + LNCaP male tumors* + LNCaP femaletumors** + PC-3 prostate carcinoma − DU-145 prostate carcinoma − BT-20breast carcinoma − T-47D breast ductal carcinoma − BT-474 breast ductalcarcinoma − MCF-7 breast adenocarcinoma − SK-BR-3 breast adenocarcinoma− MDA-MB-231 breast adenocarcinoma − LS174T colon adenocarcinoma − NIH:OVCAR-3 ovary adenocarcinoma − 3a5 hybridoma - SP2/0 fusion myeloma −placenta, human − pancreas, human − pituitary, human − liver, human −spleen, human − brain, human + heart, human − skeletal muscle, human −kidney, human − pancreas, canine − pituitary, canine − spleen, mouse −rubella (strain M33) − *LNCaP xenograft tumors propagated in maleathymic mice. **LNCaP xenograft tumors propagated in female athymicmice. + indicates detectable expression − absence of detectableexpression

Repro-PC-1.0 expression in additional carcinomas and tissues wasinvestigated by RNA blot analysis. Table 3 lists the different carcinomacell lines and human tissues that were screened for Repro-PC-1.0expression. Repro-PC-1.0 expression was hot detected in any otherprostate or non-prostate carcinoma cell line besides the LNCaP line, norwas it detected in any other normal tissue except for brain.Interestingly, this is precisely the tissue where the synaptotagminfamily is expressed, suggesting the LNCaP cells are aberrantlyexpressing a gene that encodes a protein involved in the regulatedsecretory pathway.

VI. Differential Expression of Repro-PC-1.0 and Human Synaptotagmin

In order to address whether or not Repro-PC-1.0 might represent a humansynaptotagmin isoform that was differentially expressed in LNCaP, tumorcells, we compared the expression of Repro-PC-1.0 and humansynaptotagmin in LNCaP cells, brain tissue, PC12 cells (rat adrenalmedullary cell line) and in a non-Repro-PC-1.0 or -synaptotagminexpressing human carcinoma cell line, RL95-2. RNA isolated from each ofthese sources was hybridized to either Repro-PC-1.0-specific or humansynaptotagmin-specific probes. Both forms are detectable in RNA isolatedfrom normal human brain, but only Repro-PC-1.0, and not humansynaptotagmin is expressed in LNCAP cells. Interestingly, onlyRepro-PC-1.0 sequences are detectable in PC12 cells which normallyexpress rat brain synaptotagmin I. Rat brain synaptotagmin I would nothave been detectable with either the human synaptotagmin-specific orRepro-PC-1.0-specific probes. The cross hybridization of Repro-PC-1.0 toPC12 RNA suggests that a rat homologue of Repro-PC-1.0 might also beexpressed in PC12 cells and may participate in regulated secretion. ThusRepro-PC-1.0 expression is specifically and differentially up-regulatedin LNCAP tumor cells, representing a novel human brain synaptotagminisoform that cross hybridizes with a rat homologue, distinct from ratbrain synaptotagmin I, normally expressed in PC12 cells.

VII. Discussion

Tumor-specific proteins permit insight into the mechanisms thatcontribute to the progression to malignancy. The LNCaP cell lineprovides a useful model of prostate cancer that has proven valuable inunderstanding prostate cancer. These cells were used to raise humanprostate tumor xenografts in both male and female athymic mice. Ourinitial observations of host-sex-dependent histological andmorphological differences in tumor production prompted us to investigatethese two forms of tumor as a potential source of RNA to develop asubtractive screen for identifying uniquely expressed proteins in atumor that has progressed through vascularization (angiogenesis) and hasgained metastatic character.

Tumorigenesis is often associated with either an activation of anoncogene or the inactivation of a tumor-suppressor gene (anti-oncogene).We compared the level of expression of a number of oncogenes, tumorsuppressor genes, growth factors and the androgen receptor between theirregular LNCaP tumors grown in a male environment, to LNCaP tumorsgrown in a female environment, to LNCAP cells grown in vitro, to anothernon-androgen responsive human prostate cancer cell line and to normalhuman tissue. Because tumors grown in male hosts showed a profile thatwas similar to tumors grown in females and to LNCaP cells grown invitro, we considered the two morphologically distinct tumors as asuitable source of potential message.

VIII. Fluorescent In Situ Hybridization (FISH) of Metaphase Chromosomes

Cultured lymphoblast cells are incubated with BrdU and Nacadozole for 3to 5 hours prior to harvesting with trypsin and collection bycentrifugation. The cells are resuspended in KCl and fixed in suspensionwith methanol/acetic acid on ice for 30 mm. The cell suspension isplaced on microscope slides and slowly dried at 70° C., 80% humidity. Abiotinylated cDNA probe, complementary to Repro-PC-1.0, is generatedusing standard protocols (Labeling and colorimetric detection ofnon-isotopic probes. In “Current Protocols in Molecular Biology,” F. M.Ausubel, et al., Eds., pp. 3, 18, 1-3, 18.7, John Wiley and Sons, Inc.,New York). The biotinylated probe is diluted to 3 ng/ul in ahybridization buffer containing 50% formamide, 10% dextran, 2×SSC, 50μg/ml Cot-1 DNA and incubated overnight at 37° C. with the fixed cellspread which has been preincubated with hybridization buffer. The slidesare washed twice with 0.5×SSC at 70° C., once with 4×SSC at roomtemperature, once with antibody diluent (1% BSA, 4×SSC) at roomtemperature for 5 min. and incubated for 20 min. at 37° C. withFITC-conjugated avidin diluted (10 μg/ml) in antibody diluent. Theslides are washed three times with a buffer containing 4×SSC, then witha buffer containing 4×SSC, 0.05% Tween20, then with a buffer containing4×SSC followed by counter staining for 2 min with DAPI (200 ng/ml in4×SSC) and washing with 4×SSC. The excess fluid is blotted off and theslides are mounted with a FITC stabilizing reagent containingp-phenylenediamine dihydrochloride in 90% glycerol. The hybridizationprofile is determined using a fluorescent microscope.

IX. PCR Mimic Quantitation Protocol

The following protocol is useful for amplifying Repro-PC-1.0 from a cellsample.

A. RNA Extraction:

-   -   1. Prostate is removed from patient    -   2. Within 15-30 minutes of the prostate being removed from        patient, identify normal and cancerous areas and remove        approximately 100-300 mg of tissue (approximately 1 cm×0.5        cm×0.4 cm) from each area.    -   3. If RNA is to be extracted at a later time, then place each        tissue chunk in a prelabeled piece of heavy duty aluminum foil        and immediately drop into liquid nitrogen.    -   4. Store the tissues in the −70° C. freezer.    -   5. The embedded portions are sectioned and given to the        pathologist for assessment.    -   6. The rest of the prostate is fixed in formaldehyde.    -   7. When ready for use, extract the RNA as follows:    -   8. Drop the frozen tissue into 3 ml Trizol (GIBCOBRL, catalog        number 15596-018) and homogenize using a Polytron, Tissumizer or        another adequate tissue homogenizer.    -   9. Incubate homogenized sample for 5 minutes at room        temperature.    -   10. Add 600 μl chloroform (200 μl chloroform per ml Trizol).    -   11. Mix for 15 seconds and allow to sit at room temperature for        2-3 minutes.    -   12. Spin at 12000×g for 15 minutes at 4° C.    -   13. Carefully remove the upper, colorless aqueous phase.    -   14. Precipitate the RNA using an equal volume of isopropanol.    -   15. Allow samples to sit at room temperature for 10 minutes.    -   16. Spin at 12000×g for 15 minutes at 4° C.    -   17. Remove the supernatant and wash the pellet, by breaking up        the pellet and petting up and down, with ice-cold 75% ethanol in        RNase-free water.    -   18. Spin at 12000×g for 15 minutes at 4° C.    -   19. Remove the supernatant and dry pellet in Speed-Vac for 2-3        minutes.    -   20. Resuspend the RNA in RNase-free water and incubate at 65° C.        for 10 minutes.    -   21. Take OD readings at 260 and 280 nm (for concentrated        samples, a 1:125 dilution of the sample should be used to read        the OD values. The conversion factor thus would be the A₂₆₀ of        the sample multiplied by the dilution factor of 125 multiplied        by 40 μg/ml (Sample A₂₆₀×125×40 μg/ml).        B. DNase Treatment:

The RNA samples should be DNAse I treated (GIBCOBRL, catalog number18068-015) prior to use in cDNA synthesis and PCR in order to get rid ofany contaminating genomic DNA. DNase treat the samples as follows:

-   -   1. To a microfuge tube add:

Component Sample No RT control 5 μg total RNA x μl x μl 10X DNase Ireaction buffer 1 μl 1 μl DNase I (1 unit/μl) 2 μl 1 μl DEPC water to 10μl to 10 μl

-   -   2. Incubate tubes for 15 minutes at room temperature.    -   3. Inactivate the DNase I by adding 1 μl of 25 mM EDTA.    -   4. Heat for 10 minutes at 65° C.

C. cDNA Synthesis:

Reverse transcribe RNA as follows:

-   -   1. In an microfuge tube mix the following:

Component Sample No RT control 5 μg total RNA 10 μl  10 μl  Randomhexamers (50 ng/μl) 2 μl 2 μl DEPC water 0 μl 0 μl

-   -   2. Incubate at 70° C. for 10 minutes and ice-quench for 2        minutes.    -   3. In a microfuge tube prepare the following reaction mixture in        the order indicated:

Component Per reaction add 10X PCR buffer 2 μl 25 mM MgCl₂ 2 μl 10 mMdNTP mix (10 mM each dATP, 1 μl dCTP, dGTP and dTTP) 100 mM DTT 2 μlNote* Make enough of the above mix for one extra reaction

-   -   4. Add 7 μl of the above mixture to each of the RNA/random        hexamers mixture.    -   5. Mix and centrifuge briefly.    -   6. Incubate at room temperature for 5 minutes    -   7. ADD 1 μl (200 units) of SUPERSCRIPT II RT (GIBCOBRL, catalog        number 18064-014), mix and incubate at room temperature for 10        minutes (do not add RT to No RT control tube).    -   8. Transfer tubes to 42° C. and incubate for 50 minutes.    -   9. Terminate reactions by incubating at 70° C. for 15 minutes.    -   10. Chill on ice for 2 minutes and centrifuge briefly.    -   11. Add 1 μl of RNase H (GIBCOBRL, catalog number 18021-022) and        incubate or 20 minutes at 37° C.

D. Mimic Construction:

Make the mimic dilution series as follows:

-   -   1. Label eight 0.5 ml microcentrifuge tubes 1-8.    -   2. Prepare a 100 attomole/μl stock for each of the mimics. All        dilutions should be made in mimic dilution solution (i.e. TE        containing 10 μg/ml of nucleic acid grade glycogen):        -   Prepare 20 μl of a 1:10 dilution of the 100 attomole/μl            stock and label this tube as 10 attomole/μl.        -   Add 18 μl of mimic dilution solution to tube 1 and add 2 μl            from the 10 attomole/μl tube. To make the twofold serial            Prepare 20 μl of a 1:10 dilution of the 10 attomole/μl stock            and label this tube as “1”.    -   3. Prepare the rest of the mimic stock solutions as follows:

Concentration (attomole/μl): Tube label 1 1 Add 2 μl of 10 attomole/μltube to 18 μl of mimic dilution solution, and change pipet tip 0.5 2 Add10 μl from tube 1 to 10 μl of mimic dilution solution, mix and changepipet tip 0.25 3 Add 10 μl from tube 2 to 10 μl of mimic dilutionsolution, mix and change pipet tip 0.125 4 Add 10 μl from tube 3 to 10μl of mimic dilution solution, mix and change pipet tip 0.05 5 Add 8 μlfrom tube 4 to 12 μl of mimic dilution solution, mix and change pipettip 0.025 6 Add 10 μl from tube 5 to 10 μl of mimic dilution solution,mix and change pipet tip 0.0125 7 Add 10 μl from tube 6 to 10 μl ofmimic dilution solution, mix and change pipet tip 0.005 8 Add 8 μl fromtube 4 to 12 μl of mimic dilution solution, mix and change pipet tip

Summarized in the table below are the molar and mass concentrations forthe dilution series of G3PDH (glyceraldehyde 3-phosphate dehydrogenaseand PC1:

Tube Number MIMIC PC1 1524-2156 G3PDH 71-1030 MIMIC Size 486 bp 630 bpMaster Stock Conc. ng/μl ng/μl 100 attomoles= 32 pg 42 pg cDNA Size 657bp 983 bp MIMIC Dilution Series attomoles/μl 100 32 pg/μl 42 pg/μl 103.2 pg/μl 4.2 pg/μl 1 1 320 fg/μl 420 fg/μl 2 0.5 160 fg/μl 210 fg/μl 30.25 80 fg/μl 105 fg/μl 4 0.125 40 fg/μl 52.5 fg/μl 5 0.05 16 fg/μl 21fg/μl 6 0.025 8 fg/μl 10.5 fg/μl 7 0.0125 4 fg/μl 5.25 fg/μl 8 0.005 1.6fg/μl 2.1 fg/μlIII PCR:

Set up the PCR as follows:

Volume per 25 μl Component Final concentration reaction 10X PCR buffer1X 2.5 μl 25 mM MgCl₂ 1.5 mM 1.5 μl 2.5 mM dNTP mix (mix made 200 mM 2.0μl by adding equal volumes of 10 mM dATP, dTTP, dCTP and dGTP) Upperprimer (100 ng/μl) 200 nM 0.4 μl Lower primer (100 ng/μl) 200 nM 0.4 μlMimic varies   1 μl cDNA N/A   1 μl Taq polymerase (5 U/μl) 0.05 U/μl0.25 μl  ddH₂0 N/A 15.95 μl 

-   -   1. Number the wells of a PCR plate or PCR tubes 1-8.    -   2. Add 1 μl of the mimic corresponding to the number on tube        (e.g. tube #1 gets 1 μl of mimic #1 and so on). Keep plate or        tube on ice.    -   3. Make up the following mix (make enough mix for n+1 reactions,        i.e. 9 reactions). Keep mix tube on ice:

Volume per 25 μl Volume per 9 × 25 μl Component reaction reaction 10XPCR buffer 2.5 μl 22.5 μl 25 mM MgCl₂ 1.5 μl 13.5 μl 2.5 mM dNTP mix(mix made 2.0 μl  18 μl by adding equal volumes of 10 mM dATP, dTTP,dCTP and dGTP) Upper primer (100 ng/μl) 0.4 μl 3.6 μl Lower primer (100ng/μl) 0.4 μl 3.6 μl cDNA   1 μl   9 μl Taq polymerase (5 U/μl) 0.25 μl 2.25 μl  ddH₂0 15.95 μl  143.6 μl 

-   -   4. Add 24 μl to each of the wells or tube to which the mimic had        already been added in step 2 above. Keep plate or tube on ice        while adding the mix.    -   5. Perform PCR using the following cycle:

Cycle Process Time (minutes) Temperature (° C.) Hold 1 Denaturation 5 942 Denaturation 0.5 (30 seconds) 94 3 Annealing 1 55 4 Extension 2 725-34 Repeat cycles 2-4 34 times Hold 2 Long Extension 5 72 Hold 3 Colduntil plate is 4 removed

-   -   6. Run 10 μl of the product on a 2% TAE agarose gel with 1 kb        ladder as marker.

The present invention provides novel polynucleotides encoding a prostatetumor-specific maker, Repro-PC-1.0, the protein encoded by it, andmethods for using these materials. While specific examples have beenprovided, the above description is illustrative and not restrictive.Many variations of the invention will become apparent to those skilledin the art upon review of this specification. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but instead should be determined with reference to theappended claims along with their full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted.

1. A method for use in following the progress of prostate cancer in asubject comprising the steps of: (a) detecting first and second amountsof mRNA of SEQ ID NO:1 or polypeptide of SEQ ID NO:2 in samples from thesubject at a first and a second time; and (b) comparing the first andsecond amounts, whereby an increase between the first and second amountsindicates progression of the prostate cancer and a decrease between thefirst and second amounts indicates remission of the prostate cancer.